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This work extensively studied the vasculature of mice mammary fat pads (BALB/c and C57BL/6) with special reference to haematogenous drainage routes. Mammary fat pads were five pairs (first cervical, second and third thoracic, fourth abdominal and fifth inguinal), bilaterally symmetrical, extending laterally and continuously with the subcutaneous fascia. The superficial cervical artery and vein primarily accomplished the blood vasculature of the first mammary fat pad, while the lateral thoracic and external thoracic arteries and veins supplied the second and third mammary fat pads. The superficial cervical vein (found parallel to the superficial cervical artery) drained into the external jugular vein. The lateral thoracic artery and external thoracic artery branched almost at the same level as the axillary artery (branch of subclavian artery), the latter being more medial in position. However, in some specimens, the branching of both arteries appeared to be at the same level, and their origins were indistinguishable. The lateral thoracic vein that was parallel to the lateral thoracic artery drained to the axillary vein close to the drainage of the external thoracic vein. The lateral thoracic, superficial caudal epigastric, iliolumbar and external thoracic arteries and veins vascularized the fourth mammary fat pad and displayed anastomosis among themselves. The iliolumbar vein (found parallel to the iliolumbar artery) drained into the inferior vena cava. The superficial caudal epigastric vein (found parallel to the superficial caudal epigastric artery (SCaEA)) drained into the femoral vein. Unlike humans, the internal thoracic artery and vein did not participate in the vasculature of mammary fat pads. The SCaEA and vein supplied blood and drained the fifth mammary fat pad. The anatomical continuity of the fourth and fifth mammary fat pads provided common drainage for both mammary fat pads. The BALB/c and C57BL/6 mice strains studied did not differ in topography and size of mammary fat pads. The vascular supply and drainage of the mammary fat pads also did not differ in the strains studied. Only minor variations could be noted in the small veins draining into the lateral thoracic vein. Lateral tributaries seen in the terminal end of the lateral thoracic vein were absent in the C57BL/6 mice., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Mastitis is an inflammation of mammary gland, which directly affects the milk production performance and causes huge economic losses in the dairy industry. During mastitis, the blood-milk barrier (BMB) loses its integrity and aggravates the severity of mastitis. Exogenous DNase I has been exerted protective effects in different model of tissue injury. Here, we designed a study to investigate the effects of DNase I on inflammation and BMB in a mice model of Staphylococcus aureus-induced mastitis. In the model, we found that DNase I treatment significantly alleviated the inflammatory response through decrease of inflammatory cells in mammary alveoli, MPO activity and cytokines in mammary gland. Furthermore, immunofluorescent staining and western blotting demonstrated that exogenous DNase I obviously reduced BMB permeability and changed the expression of tight junction proteins to support the re-establishment of the barrier integrity. Mechanismly, DNase I treatment inhibited NF-κB and enhanced AKT signaling pathways. Therefore, our results indicate that DNase I may be an effective treatment for attenuating mastitis., (Copyright © 2021. Published by Elsevier B.V.)

The non-neuronal cholinergic system refers to the presence of acetylcholine, choline acetyltransferase, acetylcholinesterase and cholinergic receptors, nicotinic and muscarinic (mAChRs) expressed in non-neuronal cells. The presence of mAChRs has been detected in different type of tumor cells and they are linked with tumorigenesis. We had previously documented the expression of mAChRs in murine and human mammary adenocarcinomas and the absence of these receptors in normal mammary cells of the same origins. We also demonstrated that mAChRs are involved in breast cancer progression, pointing to a main role for mAChRs as oncogenic proteins. Since the long term treatment of breast cancer cells with the muscarinic agonist carbachol promoted cell death, here we investigated the ability of low doses of this agonist combined with paclitaxel (PX), a taxane usually administered to treat breast cancer, to inhibit the progression of human MCF-7 tumor cells. We demonstrated that PX plus carbachol reduced cell viability and tumor growth in vitro probably due to a down-regulation in cancer stem cells population and in the expression of ATP "binding cassette" G2 drug extrusion pump; also a reduction in malignant-induced angiogenesis was produced by the in vivo administration of the mentioned combination in a metronomic schedule to MCF-7 tumor-bearing NUDE mice. Our results confirm that mAChRs could be considered as therapeutic targets for metronomic therapy in breast cancer as well as the usefulness of a muscarinic agonist as repositioning drug in the treatment of this type of tumors., Competing Interests: Declaration of Competing Interest There is no conflict of interest related to this study., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The objective of this study was to evaluate the association between ketonemia and serum paraoxonase-1 (PON1), malondialdehyde (MDA), and other blood components in tail and mammary veins of dairy cows. Forty-two Holstein dairy cows with decreased feed intake were divided into HIGH (≥ 1.2 mM; n = 31) and LOW (< 1.2 mM; n = 11) groups based on the β-hydroxybutyrate concentration in plasma collected from the tail vein. The HIGH group had a significantly greater plasma non-esterified fatty acid (NEFA) concentration, but significantly lower serum PON1 activity and phospholipid concentration, and a tendency to have a lower cholesterol ester concentration than the LOW group. Serum PON1 activity was not correlated with the MDA concentration but was positively correlated with serum concentrations of cholesterol esters and phospholipids, and negatively correlated with the plasma NEFA concentration. These results suggest that serum PON1 activity is reduced by hyperketonemia and the relevance of PON1 to MDA seems to not be direct, though it is involved., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

In the pig industry, labour efficiency and animal welfare have become two of the most important factors for achieving technical goals and farming competitiveness. Blood sampling is one of the most common sample-collecting techniques, but routine on-field blood collection can be very demanding for farm operators and the welfare of the animals, in particular for lactating sows. The aim of this study was to describe and investigate the mammary vein as a novel means of blood access in lactating sows that does not require coercive restraint. The study involved a total of 68 sows: 34 animals were sampled from the jugular vein (Group J) and the other 34 sows from the mammary vein (Group M). Labour time and indicators of the sow welfare (vocalizations during collection and serum cortisol concentration in the 30min after the procedure) were collected from the two groups. The total amount of labour required, calculated as the time employed to perform blood collection multiplied by the number of operators involved in performing the technique (one for Group M and two for Group J, one for restraint and one for sampling), was significantly lower in Group M than Group J (Group M, 39.83±29.45s; Group J, 82.73±55.34s; P<0.001). Mean blood volume collected at T 0 was 4.81±2.00mL and 4.84±1.73mL in Group J and Group M, respectively (P>0.05). The percentage of sows that vocalized in Group M was less than in Group J (2.94% vs. 94.12%; P<0.001). Serum cortisol concentrations were not statistically different between the two groups (P>0.05). The present study suggests greater efficiency in terms of saving labour time and reducing sow vocalization during blood collection with the use of mammary vein access compared with jugular vein access., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background : Saline is not biocompatible with saphenous vein grafts and does not protect against ischemia reperfusion injury. We compared normal heparinized saline with DuraGraft, a new graft-storage solution, in in-vitro and ex-vivo assays to evaluate the effects on cells and vascular graft tissue. Methods : Human saphenous vein (HSV) segments and isolated pig mammary veins (PMVs) were flushed and submerged in heparinized DuraGraft or heparinized saline for prespecified times. Following exposure, HSV segments were evaluated for viability and tissue morphology, and PMVs underwent histological assessments, to evaluate vein morphology and effects on the vascular endothelium. The performance of saline versus DuraGraft was compared in an ISO-compliant biocompatibility assay for cytotoxicity. Results : Loss of HSV graft-cell viability was observed as early as 15 minutes post-exposure to saline whereas viability was maintained up to 5 hours' exposure to DuraGraft. Histological analyses performed with PMVs demonstrated endothelial damage in PMVs stored in saline. Cytotoxicity assays demonstrated that saline-induced microscopically visible cell damage occurred within 60 minutes. DuraGraft-treated cells did not show evidence of damage or reactivity. Conclusions : Normal saline caused damage to vascular endothelium, loss of graft cell viability, and mediated cell damage; no evidence of damage or reactivity was observed in DuraGraft-exposed cells.

Ample research has described the assessment of dimensional changes for different teat traits, whereas diagnostic techniques to reliably assess blood circulation in teats of dairy cows are limited. Here, we describe the development and evaluation of a scanning technique to quantify blood flow in teats of dairy cows using power Doppler ultrasonography. In 2 consecutive trials, 384 teat scans [trial 1, n = 256 (sagittal plane, n = 128; transverse plane, n = 128); trial 2, n = 128 (transverse plane)] from 16 cows were obtained by the same 2 operators. Perfusion intensity from single images (trial 1) and video images (trial 2) were assessed using a commercially available software program. Intraclass correlation coefficients (ICC) and concordance correlation coefficients (CCC) were used to assess interoperator reproducibility (agreement between measurements performed by different operators) and intraoperator repeatability (agreement between measurements performed by the same operator). In trial 1, interoperator ICC and CCC indicated poor agreement (ICC ≤0.26, CCC ≤0.26). Intraoperator ICC and CCC demonstrated poor agreement between duplicate measurements within operators (ICC ≤0.19, CCC ≤0.19). Modifications after trial 1 included (1) a different ultrasound device, (2) analysis of video clips rather than single images, (3) restriction to 1 sectional plane (i.e., transverse), and (4) a scanning sequence such that repeated scans within operators were measured one after another. Through these modifications, intraoperator repeatability in trial 2 yielded fair to good agreement, with intraoperator ICC and CCC over both operators ranging from 0.44 to 0.70 and from 0.57 to 0.69, respectively, whereas interoperator ICC and CCC showed poor agreement (ICC = 0.35, CCC = 0.34). We conclude that repeatable measurements of blood perfusion intensity of teats in dairy cows can be attained with power Doppler ultrasonography. Power Doppler ultrasonography is a suitable tool to quantify slow flow in small vessels and may be an acceptable diagnostic technique to assess changes in blood circulation that result from machine milking in teats of dairy cows, although further research is necessary to validate this hypothesis., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Objectives were to investigate the effects of supplementation with corn dried distiller's grains plus solubles (DDGS) to late gestating beef cows on arterial blood flow to the mammary glands during late gestation and early lactation; colostrum and milk production; dystocia and immunity; and calf BW. Cows were fed a control (CON; n = 15; 5.1% CP; 36.2% ADF) diet consisting of 90% corn stover and 10% corn silage on a dry basis offered ad libitum or CON diet with supplementation of DDGS (0.30% of BW; SUP n = 12). Mammary gland blood flow was assessed on day 245 of gestation. At parturition, maternal and calving parameters were assessed; colostrum and jugular blood was sampled; and dams were weighed. Mammary gland blood flow and milk production was measured on day 44 of lactation. Calves were weighed fortnightly for 8 wk and at weaning. Colostrum production tended to be greater in SUP dams than in CON dams (837 vs. 614 ± 95 g, P = 0.10). Calves of SUP dams were heavier at birth and 24 h (0 h, 43.2 vs. 39.8 ± 1.0 kg, P = 0.02; 24 h, 44.0 vs. 40.4 ± 1.1 kg, P = 0.02). At birth and 24 h, blood pCO2 was greater in calves born to SUP dams (6.82 vs. 6.00 ± 0.41 kPa, P = 0.04). Serum IgG did not differ (P = 0.21) at 24 h. Ipsilateral mammary gland blood flow of SUP cows was greater than CON cows (2.76 vs. 1.76 ± 0.30 L/min; P = 0.03); however, when summed with contralateral, total blood flow was similar (P = 0.33). Hemodynamic measures on day 44 of lactation were similar (P ≥ 0.32). Milk production tended to be increased (13.5 vs. 10.2 ± 1.2 kg/d, P = 0.07) in SUP vs. CON cows. Despite similar BW through 56 d, calves from SUP cows were heavier (P = 0.04) at weaning (309.7 vs. 292.0 ± 6.0 kg). In conclusion, we accept our hypothesis that DDGS supplementation during gestation influenced mammary blood flow, milk production and calf weights. These findings implicate maternal nutrition's leverage on both nutrient and passive immunity delivery to the calf early in life as well as potential advantages on long-term performance., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The present study aimed to evaluate the mechanisms modulated by dietary arginine supplementation to sows during lactation regarding antioxidant capacity and vascularization of mammary glands. At 109 days of gestation, animals were transferred to individual farrowing crates equipped with manual feeders and automatic drinker bowls. Environmental temperature and humidity inside the farrowing rooms were registered every 15 min. At farrowing, sows were assigned in a completely randomized design to a control diet (CON) or the CON diet supplemented with 1.0% L-arginine (ARG). A total of three gilts and two sows were fed the CON diet, whereas three gilts and three sows were fed ARG diets. Sows were fed a fixed amount of 6.0 kg/day, subdivided equally in four delivery times (0700, 1000, 1300 and 1600 h) for 21 days. At weaning, sows were slaughtered and mammary tissue samples and blood from the pudendal vein were collected. Data were analyzed considering each sow as an experimental unit. Differences were considered at P&lt;0.05. L-arginine fed sows presented lower messenger RNA (mRNA) expression for prolactin receptor (P=0.002), angiopoietin1 (P=0.03) and receptor tyrosine kinase (P=0.01); higher mRNA expression for prostaglandin synthase 1 (P=0.01); a trend of decrease for glucocorticoid receptor (P=0.06) and IGF receptor 1 (P=0.07); and a trend (P=0.05) for an increased glutathione peroxidase mRNA expression. The angiopoietin2:angiopoietin1 mRNA ratio tended to increase (P=0.07) in ARG fed sows. L-arginine fed sows had greater (P=0.04) volumetric proportion of blood vessels and a trend of enhance (P=0.07) in the number of blood vessels per mm2. These findings show that 1.0% ARG supplementation to sows activates proliferative mechanisms, may improve mammary tissues' angiogenesis and tended to increase mRNA expression of genes that encode antioxidant enzymes in mammary gland of sows.

Mammary gland utilization of AA and other metabolites in response to supplemental energy from protein (PT) and supplemental energy from fat (FT) was tested in a 2 × 2 factorial arrangement using a randomized complete block design. Fifty-six Holstein-Friesian dairy cows were adapted during a 28-d control period to a basal total mixed ration consisting of 34% grass silage, 33% corn silage, 5% grass hay, and 28% concentrate on a dry matter (DM) basis. Experimental rations were fed for 28 d immediately following the control period and consisted of (1) low protein, low fat (LP/LF), (2) high protein, low fat (HP/LF), (3) low protein, high fat (LP/HF), and (4) high protein, high fat (HP/HF). To obtain the high-protein (HP) and high-fat (HF) diets, intake of the basal ration was restricted and supplemented isoenergetically [net energy (MJ/d) basis] with 2.0 kg/d rumen-protected protein (soybean + rapeseed, 50:50 mixture on a DM basis) and 0.68 kg/d hydrogenated palm fatty acids on a DM basis. Arterial and venous blood samples were collected on d 28 of both periods. Isoenergetic supplements (MJ/d) of protein and fat independently and additively increased milk yield, PT increased protein yield, and FT increased fat yield. A PT × FT interaction affected arterial concentration of all essential AA (EAA) groups, where they increased in response to PT by a greater magnitude at the LF level (on average 35%) compared with the HF level (on average 14%). Mammary gland plasma flow was unaffected by PT or FT. Supplementation with PT tended to decrease mammary clearance of total EAA and decreased group 1 AA clearance by 19%. In response to PT, mammary uptake of total EAA and group 2 AA increased 12 and 14%, respectively, with significantly higher uptake of Arg, Ile, and Leu. Energy from fat had no effect on mammary clearance or uptake of any AA group. The mammary gland uptake:milk protein output ratio was not affected by FT, whereas PT increased this ratio for EAA and group 2 AA. Arterial plasma insulin concentration decreased in response to FT, in particular on the HP/HF diet, as indicated by a PT × FT interaction. Arterial concentrations of nonesterified fatty acids, triacylglycerol, and long-chain fatty acids increased in response to FT, and concentrations of β-hydroxybutyrate and acetate decreased in response to FT only at the HP level. Mammary clearance and uptake of triacylglycerol and long-chain fatty acids increased in response to FT. Energy from PT and FT increased lactose yield despite no change in arterial glucose concentration or mammary glucose uptake. Mammary-sequestered glucose with PT or FT was used in the same amount for lactose synthesis, and a positive net mammary glucose balance was found across all treatments. Results presented here illustrate metabolic flexibility of the mammary gland in its use of aminogenic versus lipogenic substrates for milk synthesis., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The aim of the research reported in this Research Communication was to identify differentially expressed proteins in dairy cows with normal and lutein diet and to elucidate the mechanisms of lutein-induced effects on bovine mammary gland metabolism using a comparative proteomic approach. Thirty-three differentially expressed proteins were identified from mammary gland of control diet-fed and lutein diet-fed dairy cows. Among these proteins, 15 were upregulated and 18 were downregulated in the lutein group. Functional analysis of the differentially expressed proteins showed that increased blood flow, depressed glycolysis, enhanced lactose anabolism, decreased fatty acid oxidation and up-regulated beta lactoglobulin expression were connected with lutein addition. These results suggested that the increased blood flow, reduced glucose catabolism, enhanced capacity for milk lactose synthesis, depressed fatty acid catabolism and increased expression of antioxidantion related protein may be the prime factors contributing to the increased milk production and enhanced immune status in lutein-fed dairy cows. This study provides molecular mechanism of dietary lutein in regulating lactation of dairy cows.

Fluorescence imaging of biological systems in the second near-infrared (NIR-II, 1000-1700 nm) window has shown promise of high spatial resolution, low background, and deep tissue penetration owing to low autofluorescence and suppressed scattering of long wavelength photons. Here we develop a bright organic nanofluorophore (named p-FE) for high-performance biological imaging in the NIR-II window. The bright NIR-II >1100 nm fluorescence emission from p-FE affords non-invasive in vivo tracking of blood flow in mouse brain vessels. Excitingly, p-FE enables one-photon based, three-dimensional (3D) confocal imaging of vasculatures in fixed mouse brain tissue with a layer-by-layer imaging depth up to ~1.3 mm and sub-10 µm high spatial resolution. We also perform in vivo two-color fluorescence imaging in the NIR-II window by utilizing p-FE as a vasculature imaging agent emitting between 1100 and 1300 nm and single-walled carbon nanotubes (CNTs) emitting above 1500 nm to highlight tumors in mice.

Dairy milk consists of more than 85% water. Therefore, understanding the regulation of fluid absorption in the mammary gland is relevant to improving milk production. In recent decades, studies using different approaches, including blood flow, transmembrane fluid flow, tight junction, fluid flow of the paracellular pathway and functional mammary epithelial cell state, have been conducted aiming to investigate how mammary gland fluid absorption is regulated. However, the relationship between regulation mechanisms of fluid flow and milk production has not been studied systematically. The present review summarizes a series of key milk yield regulatory factors mediated by whole-mammary fluid flow, including milk, mammary blood flow, blood/tissue fluid-cell fluid flow and cell-alveolus fluid flow. Whole-mammary fluid flow regulates milk production by altering transporter activity, ion channels, local microcirculation-related factors, driving force of fluid transport (osmotic pressure or electrochemical gradient), cellular connection state and a cell volume sensitive mechanism. In addition, whole-mammary fluid flow plays important roles in milk synthesis and secretion. Knowledge gained from fluid flow-mediated regulatory mechanisms of the dairy mammary gland will lead to a fundamental understanding of lactation biology and will be beneficial for the improvement of dairy productivity. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

During lactation, mammary epithelial cells (MECs) form the blood-milk barrier by less-permeable tight junctions (TJs) to prevent the leakage of milk components. Phytoestrogens affect the proliferation, differentiation, and apoptosis of MECs. However, it remains unclear whether phytoestrogens are involved in the blood-milk barrier. Therefore, we investigated the influence of phytoestrogens (coumestrol, genistein, and daidzein) by using an in vitro mouse-MEC-culture model. The results showed that coumestrol and genistein changed the expression of TJ proteins (claudins-3 and -4 and occludin), weakened barrier function, and reduced β-casein production. Daidzein also weakened barrier function without inhibiting β-casein production. Additionally, coumestrol and genistein induced apoptosis in MECs. These results indicate that phytoestrogens weaken the blood-milk barrier by directly affecting TJs and the cellular viability of lactating MECs in different ways.

The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Positive effects have been observed in offspring from beef cows supplemented with corn dried distillers grain with solubles (DDGS) during late gestation. The hypothesis of this study was that late gestational DDGS supplementation to beef cows would increase blood flow (BF) to the gravid uterus and mammary gland thus impacting birthweight and post-natal growth of the offspring. Experiment 1 investigated mammary gland BF in multiparous cows during late pregnancy. Beef cows were fed a control (CON1) diet of low-quality hay (n = 5) or a supplement diet (SUP1) of low-quality hay with DDGS [1.7 g/kg of body weight (BW); n = 6]. In Experiment 2, multiparous late pregnant beef cows were fed either a control (CON2) diet of a low-quality hay (n = 4) or a supplement diet (SUP2) of low-quality hay with DDGS (1.7 g/kg of BW; n = 5). Uterine and mammary gland BF were recorded every 21 days during late gestation. In Experiment 1, there were no effects of diet or day on mammary gland hemodynamics. In Experiment 2, total and ipsilateral uterine BF was less (p ≤ 0.04) in SUP2 vs. CON2 cows and similar BF to contralateral horns. Mammary gland BF was unaltered by maternal supplementation. Even when measured in two different years in two different environments, mammary gland BF remained unaltered to DDGS supplementation. Investigations on the mechanism that may impact uterine BF during late gestation remain to be known., (Journal of Animal Physiology and Animal Nutrition © 2016 Blackwell Verlag GmbH.)

To investigate responses of milk protein synthesis and mammary AA metabolism to a graded decrease of postruminal Lys supply, 4 lactating goats fitted with jugular vein, mammary vein, and carotid artery catheters and transonic blood flow detectors on the external pudic artery were used in a 4 × 4 Latin square experiment. Goats were fasted for 24 h and then received a 9-h intravenous infusion of an AA mixture plus glucose. Milk yield was recorded and samples were taken in h 2 to 8 of the infusion period; a mammary biopsy was performed in the last hour. Treatments were graded decrease of lysine content in the infusate to 100 (complete), 60, 30, or 0% as in casein. Lysine-removed infusions linearly decreased milk yield, tended to decrease lactose yield, and tended to increase milk fat to protein ratio. Milk protein content and yield were linearly decreased by graded Lys deficiency. Mammary Lys uptake was concomitantly decreased, but linear regression analysis found no significant relationship between mammary Lys uptake and milk protein yield. Treatments had no effects on phosphorylation levels of the downstream proteins measured in the mammalian target or rapamycin pathway except for a tended quadratic effect on that of eukaryotic initiation factor 2, which was increased and then decreased by graded Lys deficiency. Removal of Lys from the infusate linearly increased circulating glucagon and glucose. Removal of Lys from the infusate linearly decreased arterial and venous concentrations of Lys. Treatments also had a significant quadratic effect on venous Lys, suggesting mechanisms to stabilize circulating Lys at a certain range. The 2 infusions partially removing Lys resulted in a similar 20% decrease, whereas the 0% Lys infusion resulted in an abrupt 70% decrease in mammary Lys uptake compared with that of the full-AA mixture infusion. Consistent with the abrupt decrease, mammary Lys uptake-to-output ratio decreased from 2.2 to 0.92, suggesting catabolism of Lys in the mammary gland could be completely prevented when the animal faced severe Lys deficiency. Mammary blood flow was linearly increased, consistent with the linearly increased circulating nitric oxide by graded Lys deficiency, indicating mechanisms to ensure the priority of the mammary gland in acquiring AA for milk protein synthesis. Infusions with Lys removed increased mammary clearance rate of Lys numerically by 2 to 3 fold. In conclusion, the decreased milk protein yield by graded Lys deficiency was mainly a result of the varied physiological status, as indicated by the elevated circulating glucagon and glucose, rather than a result of the decreased mammary Lys uptake or depressed signals in the mTOR pathway. Mechanisms of Lys deficiency to promote glucagon secretion and mammary blood flow and glucagon to depress milk protein synthesis need to be clarified by future studies., (Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The objectives of the present study were to design a method to estimate mammary plasma flow (MPF) in lactating sows using downstream dilution of -aminohippuric acid (AH) and to compare these estimates with MPF estimates based on specific AA as internal markers (MPF-AA). A permanent indwelling catheter was surgically implanted in the femoral artery, and another 2 were inserted in the right cranial mammary vein of 8 second- and third-parity sows on d 76 ± 2 SEM of gestation. On the 3rd and 17th days in milk, arterial and venous blood samples were drawn in hourly intervals from 0.5 h before until 6.5 h after feeding. The MPF in the right cranial mammary vein was measured by downstream dilution of infused AH (3.0 mmol/h). Total MPF-AH was calculated assuming that the measured flow constituted the flow from 5 out of 14 suckled glands on the basis of the anatomical structure of the mammary vascular system. Total MPF-AA was estimated on the basis of the output of the specific AA marker in milk and the arteriovenous differences of the marker as free AA in plasma, assuming a direct transfer of AA from plasma to milk protein. Total MPF-AH was 6,860 L/d in early lactation and increased to 8,953 L/d at peak lactation ( = 0.003). In early lactation, MPF-AA estimates were greater or tended to be greater (132% to 175%; < 0.10) than MPF-AH estimates for all internal markers, except Met (119%). Moreover, MPF-AH was correlated with MPF-AA only for MET as an internal marker ( = 0.74; = 0.03) in early lactation. In contrast, MPF-AH and MPF-AA estimates did not differ and were well correlated at peak lactation with the strongest correlation observed when Met ( = 0.84; = 0.009) and Phe + Tyr ( = 0.82; = 0.01) were used as the internal AA markers. Litter gain increased from d 3 to 17 of lactation (2.13 vs. 3.46 g/d; = 0.001) and was correlated with MPF-AH during lactation ( = 0.74; < 0.001), whereas no correlation between litter gain and MPF-AA was observed ( > 0.10). These results suggest that downstream dilution of infused AH and the AA methods are applicable methods to estimate MPF at peak lactation. The reason for the observed discrepancy in early lactation between MPF- AH and MPF-AA is not obvious but might be related to the rapid metabolic changes observed in early lactation. In conclusion, MPF measured by downstream dilution of mammary infused AH was higher at peak compared to early lactation, which the internal AA marker approach failed to show.

The objective was to determine the blood flow variables in the uterine arteries and the pudendoepigastric trunks, which supply the mammary gland, and relate these variables to the occurrence of uterine disease, milk yield, and pregnancy result. To achieve this, 119 multiparous German Holstein cows were examined using color Doppler sonography once during the dry period and on Days 7, 14, 28, 42, 56, 66, 76, 86, and 96 postpartum (pp). Cows with retained fetal membranes or metritis had a higher blood flow volume and time-averaged maximum velocity and a lower pulsatility index in the uterine arteries on Days 7, 14 and 28 pp (P < 0.05). Milk yield was correlated with blood flow volume in the pudendoepigastric trunks on examination Days 7 to 96 pp with the exception of Day 76 (P < 0.05), and with time-averaged maximum velocity on Days 7 and 14 pp (P < 0.05). The pulsatility index was greater in the left pudendoepigastric trunk on examination Days 7 to 76 pp than in the right pudendoepigastric trunk (P < 0.05). Milk yield did not affect pregnancy result and was not related to uterine perfusion. Increased uterine perfusion in cows with retained fetal membrane and metritis may be due to increased uterine size attributable to delayed involution. High mammary perfusion in high-yielding cows is due to an increased demand for nutrients and oxygen. Color Doppler sonography is a useful method for the investigation of the effect of uterine disease on uterine blood flow and of the effect of milk yield on mammary perfusion., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A mechanistic model that predicts sensible and latent heat fluxes from the udder of a dairy cow was developed. The prediction of the model was spot validated against measured data from the literature, and the result agreed within 7% of the measured value for the same ambient temperature. A dairy cow can lose a significant amount of heat (388W/m(2)) from the udder. This suggests that the udder could be considered as a heat sink. The temperature profile through the udder tissue (core to skin) approached the core temperature for an air temperature ≥37°C whereas the profile decreased linearly from the core to skin surface for an air temperature less than 37°C. Sensible heat loss was dominant when ambient air temperature was less than 37.5°C but latent heat loss was greater than sensible heat loss when air temperature was ≥37.5°C. The udder could lose a total (sensible + latent) heat flux of 338W/m(2) at an ambient temperature of 35°C and blood-flow rate of 3.2×10(-3)m(3)/(sm(3) tissue). The results of this study suggests that, in time of heat stress, a dairy cow could be cooled by cooling the udder only (e.g., using an evaporative cooling jacket)., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Efficient milk protein synthesis requires that the essential AA be presented to the mammary gland in the right amount and proportion to maximize protein synthesis and minimize losses. This study investigated the effects of individual AA deficiencies on cow productivity, mammary metabolism, and glucose whole-body rate of appearance. Five Holstein cows were used in a 5 × 5 Latin square design trial with 10-d periods. Treatments were abomasal infusions of (1) water (CTL); (2) complete AA mixture (TAA); (3) TAA without Phe (No-Phe); (4) TAA without Thr (No-Thr); and (5) TAA without Trp (No-Trp). Each treatment was compared with TAA. Treatment did not affect milk, fat, or lactose yields. Arterial concentrations of Phe, Thr, and Trp decreased with their respective deletions by 60, 76, and 69%. In response to the decreased arterial supply of the deleted AA, mammary plasma flow significantly increased by 55% with No-Thr but did not increase with No-Phe or No-Trp. Mammary uptake of Phe was reduced by No-Phe, accompanied by a reduced milk protein yield; uptakes of Thr and Trp were not affected by their respective deletions, and milk protein yield did not decrease with these treatments. Deletion of Phe tended to reduce its mammary uptake relative to milk output (U:O), accompanied by an increased U:O of Tyr, but deletion of Thr and Trp did not affect the U:O of the corresponding AA. Plasma urea-N concentration was lower with CTL and tended to be higher with No-Phe. Arterial concentrations and mammary uptake of acetate, β-hydroxybutyrate, glucose, and lactate were unaffected by treatment. Treatment had no effect on glucose rate of appearance at the whole-body level. Lactose output as a percentage of glucose whole-body rate of appearance was not affected by treatment. Overall, the study indicated that a deficiency of Phe negatively affected productivity and mammary metabolism but that a deficiency of Thr or Trp did not., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Previous work shows that mammary uptake of milk precursors from blood can be affected by the rate of blood flow (F) to the glands. The purpose of the current work was to test the ability of compartmental and cylindrical capillary models to account for the variation in mammary extraction and net uptake of plasma metabolites produced by perturbation of mammary F. The data for model fitting were obtained from a previous experiment in which mammary arteriovenous differences of acetate + β-hydroxybutyrate (2C), glucose, triacylglycerol (TAG), and long-chain fatty acids (LCFA) were measured in 4 cows before, during, and after intraarterial infusion of inhibitors of endothelial nitric oxide synthase and cyclooxygenase, which are 2 major systems of F control in the mammary glands. The 4 models tested were (1) constant extraction within each cow, (2) clearance from an extracellular compartment is a linear function of F with an intercept, (3) total capillary volume in a cylindrical representation is a linear function of F with an intercept, and (4) uptake from an extracellular compartment obeys Henri-Michaelis-Menten kinetics, where maximum velocity (Vmax) is a linear function of F with an intercept. According to prediction errors, model 4 fitted 2C extraction data best, accounting for 82% of the observed variation. The estimated Km (Henri-Michaelis-Menten constant) for venous 2C was 0.4 mM. For glucose clearance, a variant of model 2 with a positive effect of 2C uptake on clearance was identified as best, producing a coefficient of determination (R(2)) of 0.31. For TAG, model 2 with a positive effect of arterial TAG concentration on TAG clearance was best, with an R(2) of 0.22. For LCFA, model 2 with a positive effect of arterial LCFA on LCFA clearance was best, with an R(2) of 0.29. Models 2 and 3 fitted the extraction data with the same R(2)-values and prediction errors, so both compartmental and cylindrical approaches to describing the vascular bed were equally capable of describing the effect of F on mammary uptakes. A combined fit of all best-fit models to extraction data for all 4 metabolites at once explained 52, 42, 73, and 77% of variation in net uptakes of 2C, glucose, TAG, and LCFA, respectively. According to the fitted model, each 1 L/min increase in F increased the mammary volumes of distribution of 2C, glucose, TAG, and LCFA by 13, 14, 18, and 7%, respectively., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The vascular network within the developing mammary gland (MG) grows in concert with the epithelium to prepare for lactation, although the mechanisms coordinating this vascular development are unresolved. Vascular endothelial growth factor A (VEGF-A) mediates angiogenesis and vascular permeability in the MG during pregnancy and lactation, where its expression is upregulated by prolactin. Given our previous finding that late-gestational hyperprolactinemia induced by domperidone (DOM) increased subsequent milk yield from gilts, we sought to establish changes in vascular development during late gestation and lactation in the MGs of these pigs and determine whether DOM altered MG angiogenesis and the factors regulating it. Gilts received either no treatment (n = 6) or DOM (n = 6) during late gestation, then had their MG biopsied from late gestation through lactation to assess microvessel density, VEGF-A distribution and messenger RNA expression, and aquaporin (AQP) gene expression. Microvessel density in the MG was unchanged during gestation then increased between days 2 and 21 of lactation (P < 0.05). The local expression of messenger RNA for VEGF-A120, VEGF-A147, VEGF-A164, VEGF-A164b, VEGF-A188, VEGF receptors-1 and -2, and AQP1 and AQP3 all generally increased during the transition from gestation to lactation (P < 0.05). Immunostaining localized VEGF-A to the apical cytoplasm of secretory epithelial cells, consistent with a far greater concentration of VEGF-A in colostrum and/or milk vs plasma (P < 0.0001). There was no effect of DOM on any of the variables analyzed. In summary, we found that vascular development in the MG increases during lactation in first-parity gilts and that VEGF-A is a part of the mammary secretome. Although late-gestational hyperprolactinemia increases milk yield, there was no evidence that it altered vascular development., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective of the study was to record, by means of ultrasonographic examination, changes occurring during lactogenesis in the udder of healthy ewes and of ewes with pregnancy toxaemia. The work was carried out in 28 ewes, 16 with pregnancy toxaemia (group A) and 12 healthy controls (group B). B-mode and Doppler ultrasonographic examination of the udder of ewes was performed. During the last month of pregnancy, grey-scale intensity values of mammary parenchyma in group A were significantly greater than in group B (P = 0.007), as was also the progressive increase in grey-scale intensity values in both groups (P < 0.001). Blood mammary input was significantly greater in ewes of group B than in ewes of group A (P < 0.05), as was also the progressive increase in blood input in both groups (P < 0.001). Further, differences between the two groups were identified in pulsatility index (P = 0.007) and in mean blood velocity (P = 0.036), but only during the last fortnight of pregnancy. After lambing, grey-scale values decreased sharply compared to those in pregnancy (P < 0.01), whilst blood input, pulsatility index and mean blood velocity continued the same trend as at the last stage of pregnancy, with differences between the two groups still prevalent (P < 0.05). There was a reverse correlation between grey-scale intensity values and milk quantities (P < 0.035) and a correlation between blood input and milk quantities (P < 0.07). The progressive increase in the diameter of the external pudendal artery was significant (P < 0.001), but no significant differences were evident between the two groups (P > 0.35). Differences between group A and group B in all other haemodynamic parameters studied were not significant, neither throughout the last month of pregnancy (P > 0.25), nor during the first week of lactation (P > 0.06). However, their progressive changes during the last month of pregnancy were significant (P < 0.02).

As the demand for food increases with exponential growth in the world population, it is imperative that we understand how to make livestock production as efficient as possible in the face of decreasing available natural resources. Moreover, it is important that livestock are able to meet their metabolic demands and supply adequate nutrition to developing offspring both during pregnancy and lactation. Specific nutrient supplementation programs that are designed to offset deficiencies, enhance efficiency, and improve nutrient supply during pregnancy can alter tissue vascular responses, fetal growth, and postnatal offspring outcomes. This review outlines how vascularity in nutrient transferring tissues, namely the maternal gastrointestinal tract, the utero-placental tissue, and the mammary gland, respond to differing nutritional planes and other specific nutrient supplementation regimes.

The objective of this study was to clarify the effect of different milking frequencies under an automatic milking system (AMS) on milk yield, plasma metabolite profiles and mammary arterial-venous (A-V) differences of milk precursors by mammary tissues in early lactation cows. Twelve Holstein cows were divided into two and four times milking frequency treatments by AMS after calving to 50 days postpartum. Cows were given a partial mixed ration ad libitum and a concentrate diet at every milking. Dry matter intake increased similarly in both treatments with advancing postpartum days. Milk yield was greater (P < 0.001) by 25% with four times milking, but milk composition was not affected by milking frequency. Body weight change was also not affected by milking frequency. Arterial concentrations of glucose and glutamate were lower (P < 0.05) for four times milking frequency. However, arterial concentration of nonesterified fatty acids did not differ between treatments. Although mammary A-V differences of plasma concentration for most milk precursors did not differ between treatments, estimated plasma flow was higher (P < 0.05) for four times milking frequency. These results indicate that higher milking frequency may increase mammary uptake of milk precursors, whereas may not affect the extent of fat mobilization of early lactating cows from day 20 postpartum onward., (© 2014 Japanese Society of Animal Science.)

To test the effect of mammary blood flow on net uptakes of milk precursors by the mammary glands, inhibitors of nitric oxide synthase (NOS) and cyclooxygenase (COX) were infused into the mammary circulation of 4 lactating cows. Inhibitors were infused in a 4×4 Latin square design, where treatments were infusion for 1 h of saline, NOS inhibitor (Nω-nitro-l-arginine methyl ester hydrochloride), COX inhibitor (indomethacin), or both NOS + COX inhibitors into one external iliac artery. Para-aminohippuric acid was also infused to allow for estimation of iliac plasma flow (IPF), of which approximately 80% flows to the mammary glands. Blood samples were collected before, during, and after inhibitor infusion from the contralateral external iliac artery and ipsilateral mammary vein. Inhibition of COX and NOS each produced a decrease in IPF, although the NOS effect was smaller and IPF continued to be depressed throughout the recovery period. The combination of COX and NOS inhibition produced a 50% depression in IPF and there was no carryover into the recovery period. Treatments that depressed IPF also increased arterial concentrations of acetate, β-hydroxybutyrate (BHBA), and glucose. Similarly, arteriovenous differences of acetate, BHBA, and glucose were all increased during IPF depression. To correct for a potential effect of arterial concentration, arteriovenous differences were normalized to arterial concentration, producing an extraction percentage. Inhibition of COX increased glucose extraction and tended to increase acetate and BHBA extraction. Dual inhibition only increased BHBA extraction and had no effect on mammary extraction of other metabolites. These extractions did not increase because clearances of glucose and TAG decreased as IPF decreased, and clearances of acetate and BHBA tended to decrease. Net uptake of TAG was depressed by dual NOS/COX inhibition, whereas uptakes of acetate, BHBA, and glucose were not affected by any of the treatments. To separate effects of flow from effects of arterial concentration, uptakes were regressed against IPF and arterial concentration simultaneously. According to the slopes of the regressions, a 10% decrease in IPF from the mean observed during saline infusion resulted in 3.8, 7.3, and 10.4% decreases in uptakes of acetate, glucose, and triacylglycerol, respectively. These findings indicate that mammary blood flow affects milk precursor uptake, and that clearance should not be assumed constant to predict mammary uptakes of milk precursors in situations where blood flow is changing., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

This study aimed to evaluate the sonographic characteristics of the udder and teats and to determine the Doppler indexes of mammary artery in healthy and undergoing subclinical and clinical mastitis goats. Thirty animals among Saanen and Alpine Brown goats were arranged in three groups, healthy goats (HG), goats with subclinical mastitis (SMG) and goats with clinical mastitis (CMG). Using the B-mode, the sonographic characteristics (echotexture and echogenicity) and biometry (diameter and area of the udder cistern, diameter and area of the teat cistern and thickness of the teat wall) were evaluated. Using Doppler ultrasonography, the vascular indexes of the mammary artery were obtained. It was observed hyperechogenicity with solid component in the gland cistern when comparing animals with clinical mastitis and healthy mammary tissue. Regarding the echotexture of the breast tissue, there was heterogeneity in the mammary parenchyma on the three groups, for the milk, it was observed homogeneity for animals on HG and SMG and heterogeneity for animals on CMG. Grey-scale quantitative assessment revealed increase in echogenicity (mean value) for all the structures when comparing the three groups. Biometry did not reveal statistical difference between groups, for none of the evaluated structures. Doppler examination of the mammary artery showed the decrease of end diastolic velocity and raise of pulsatility index between groups. The association of B-mode and Doppler ultrasonography is useful for the evaluation of the udder of dairy goats with mastitis. It is a sensitive and specific method for the study of this disease. Doppler mode was unable to establish reliable criteria for diagnosis of subclinical mastitis. Moreover, the quantification of echogenicity is a useful technique for the evaluation of the milk in animals with mastitis; therefore, it is suggested that it can be used as complementary technique for the diagnosis of mastitis in goats., (© 2015 Blackwell Verlag GmbH.)

Vasoinhibins are a family of peptides that act on endothelial cells to suppress angiogenesis and promote apoptosis-mediated vascular regression. Vasoinhibins include the N-terminal fragments from prolactin (PRL), GH, and placental lactogen. One of the vasoinhibins, the N-terminal PRL fragment of 16 kDa, is generated by the lysosomal representative protease cathepsin D (Cath D). Because the normal growth and involution of the mammary gland (MG) are profoundly affected by the expansion and regression of blood vessels and also because PRL stimulates the growth and differentiation of MG, we proposed that intact PRL produced during lactation contributes to MG angiogenesis and increased blood flow, whereas during involution, the N-terminal PRL fragment would have proapoptotic effects on mammary epithelial cells (MECs). Therefore, we investigated the production of the N-terminal PRL fragment and its direct effect on the MG. Mouse PRL (mPRL) was proteolytically cleaved by Cath D between amino acids 148 and 149. N-terminal PRL fragment and Cath D expression increased during MG involution. Furthermore, incubation of MG fragments and MCF7 with recombinant 16 kDa mPRL revealed a proapoptotic effect in MECs. Ectopic mPRL in MECs was cleaved to 16 kDa PRL by Cath D in the MG lysosomal fraction. The majority of PRL derived from pituitary gland was cleaved to 16 kDa PRL in culture medium. Therefore, N-terminal PRL fragment increases during the involution period, has a proapoptotic effect on MECs, and is mainly generated by secreted Cath D in the extracellular space of MG.

Two trials were conducted to examine factors potentially influencing the measurement of blood β-hydroxybutyric acid (BHBA) in dairy cows. The objective of the first trial was to study effects of sampling time on BHBA concentration in continuously fed dairy cows. Furthermore, we determined test characteristics of a single BHBA measurement at a random time of the day to diagnose subclinical ketosis considering commonly used cut-points (1.2 and 1.4 mmol/L). Finally, we set out to evaluate if test characteristics could be enhanced by repeating measurements after different time intervals. During 4 herd visits, a total of 128 cows (8 to 28 d in milk) fed 10 times daily were screened at 0900 h and preselected by BHBA concentration. Blood samples were drawn from the tail vessels and BHBA concentrations were measured using an electronic BHBA meter (Precision Xceed, Abbott Diabetes Care Ltd., Witney, UK). Cows with BHBA concentrations ≥0.8 mmol/L at this time were enrolled in the trial (n=92). Subsequent BHBA measurements took place every 3h for a total of 8 measurements during 24 h. The effect of sampling time on BHBA concentrations was tested in a repeated-measures ANOVA repeating sampling time. Sampling time did not affect BHBA concentrations in continuously fed dairy cows. Defining the average daily BHBA concentration calculated from the 8 measurements as the gold standard, a single measurement at a random time of the day to diagnose subclinical ketosis had a sensitivity of 0.90 or 0.89 at the 2 BHBA cut-points (1.2 and 1.4 mmol/L). Specificity was 0.88 or 0.90 using the same cut-points. Repeating measurements after different time intervals improved test characteristics only slightly. In the second experiment, we compared BHBA concentrations of samples drawn from 3 different blood sampling locations (tail vessels, jugular vein, and mammary vein) of 116 lactating dairy cows. Concentrations of BHBA differed in samples from the 3 sampling locations. Mean BHBA concentration was 0.3 mmol/L lower when measured in the mammary vein compared with the jugular vein and 0.4 mmol/L lower in the mammary vein compared with the tail vessels. We conclude that to measure BHBA, blood samples of continuously fed dairy cows can be drawn at any time of the day. A single measurement provides very good test characteristics for on-farm conditions. Blood samples for BHBA measurement should be drawn from the jugular vein or tail vessels; the mammary vein should not be used for this purpose., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

To test which, if any, of the major milk precursors can elicit a rapid change in the rate of mammary blood flow (MBF) and to define the time course and magnitude of such changes, 4 lactating cows were infused with glucose, amino acids, or triacylglycerol into the external iliac artery feeding one udder half while iliac plasma flow (IPF) was monitored continuously by dye dilution. Adenosine and saline were infused as positive and negative controls, respectively, and insulin was infused to characterize the response to a centrally produced anabolic hormone. To test the roles of cyclooxygenase, NO synthase and ATP-sensitive K (KATP) channels in nutrient-mediated changes in blood flow, their respective inhibitors-indomethacin, Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME), and glibenclamide-were infused simultaneously with glucose. Each day, 1 infusate was given twice to each cow, over a 20-min period each time, separated by a 20-min washout period. In addition, each treatment protocol was administered on 2 separate days. A 73% increase in IPF during adenosine infusion showed that the mammary vasodilatory response was quadratic in time, with most changes occurring in the first 5min. Glucose infusion decreased IPF by 9% in a quadratic manner, most rapidly in the first 5min, indicating that a feedback mechanism of local blood flow control, likely through adenosine release, was operative in the mammary vasculature. Amino acid infusion increased IPF 9% in a linear manner, suggesting that mammary ATP utilization was stimulated more than ATP production. This could reflect a stimulation of protein synthesis. Triacylglycerol only tended to decrease IPF and insulin did not affect IPF. A lack of IPF response to glibenclamide indicates that KATP channels are not involved in MBF regulation. Indomethacin and L-NAME both depressed IPF. In the presence of indomethacin, glucose infusion caused a quadratic 9% increase in IPF. Indomethacin is an inhibitor of mitochondrial function, so the glucose-induced increase in IPF was interpreted as feedback on mammary adenosine release from an anabolic response to glucose. Because NO synthase was not inhibited during indomethacin infusion, the feedback system is postulated to act through endothelial NO synthase. In the presence of L-NAME, glucose infusion had no effect on IPF, indicating that endothelial cyclooxygenase is not involved in glucose-induced changes in MBF., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Mesenchymal stromal cells (MSCs) are multipotent adult stem cells which are recruited to the tumor microenvironment (TME) and influence tumor progression through multiple mechanisms. In this study, we examined the effects of MSCs on the tunmorigenic capacity of 4T1 murine mammary cancer cells. It was found that MSC-conditioned medium increased the proliferation, migration, and efficiency of mammosphere formation of 4T1 cells in vitro. When co-injected with MSCs into the mouse mammary fat pad, 4T1 cells showed enhanced tumor growth and generated increased spontaneous lung metastasis. Using in vivo fluorescence color-coded imaging, the interaction between GFP-expressing MSCs and RFP-expressing 4T1 cells was monitored. As few as five 4T1 cells could give rise to tumor formation when co-injected with MSCs into the mouse mammary fat pad, but no tumor was formed when five or ten 4T1 cells were implanted alone. The elevation of tumorigenic potential was further supported by gene expression analysis, which showed that when 4T1 cells were in contact with MSCs, several oncogenes, cancer markers, and tumor promoters were upregulated. Moreover, in vivo longitudinal fluorescence imaging of tumorigenesis revealed that MSCs created a vascularized environment which enhances the ability of 4T1 cells to colonize and proliferate. In conclusion, this study demonstrates that the promotion of mammary cancer progression by MSCs was achieved through the generation of a cancer-enhancing microenvironment to increase tumorigenic potential. These findings also suggest the potential risk of enhancing tumor progression in clinical cell therapy using MSCs. Attention has to be paid to patients with high risk of breast cancer when considering cell therapy with MSCs.

Photoacoustic (PA) imaging for biomedical applications has been under development for many years. Based on the many advances over the past decade, a new photoacoustic imaging system has been integrated into a micro-ultrasound platform for co-registered PA-ultrasound (US) imaging. The design and implementation of the new scanner is described and its performance quantified. Beamforming techniques and signal processing are described, in conjunction with in vivo PA images of normal subcutaneous mouse tissue and selected tumor models. In particular, the use of the system to estimate the spatial distribution of oxygen saturation (sO2) in blood and co-registered with B-mode images of the surrounding anatomy are investigated. The system was validated in vivo against a complementary technique for measuring partial pressure of oxygen in blood (pO2). The pO2 estimates were converted to sO2 values based on a standard dissociation curve found in the literature. Preliminary studies of oxygenation effects were performed in a mouse model of breast cancer (MDA-MB-231) in which control mice were compared with mice treated with a targeted antiangiogenic agent over a 3 d period. Treated mice exhibited a >90% decrease in blood volume, an 85% reduction in blood wash-in rate, and a 60% decrease in relative tissue oxygenation.

Objectives: Tumour xenografts of well-discernible sizes can be examined well by molecular ultrasound. Here, we investigated whether very early breast carcinomas express sufficient levels of VEGFR2 for reliable molecular ultrasound imaging with targeted microbubbles., Methods: MCF-7 breast cancer xenografts were orthotopically implanted in nude mice (n = 26). Tumours measuring from 4 mm(3) (2 mm diameter) up to 65 mm(3) (5 mm diameter) were examined with automated 3D molecular ultrasound using clinically translatable VEGFR2-targeted microbubbles (BR55). Additionally, the relative tumour blood volume was assessed with non-targeted microbubbles (BR38). In vivo ultrasound data were validated by quantitative immunohistochemistry., Results: Very small lesions 2 mm in diameter showed the highest binding of VEGFR2-specific microbubbles. In larger tumours significantly less BR55 accumulated (p = 0.023). Nonetheless, binding of VEGFR2-targeted microbubbles was still high enough for imaging. The relative blood volume was comparable at all tumour sizes. Both findings were confirmed by immunohistochemistry. Additionally, a significantly enhanced number of large and mature vessels were detected with increasing tumour size (p < 0.01), explaining the decrease in VEGFR2 expression during tumour growth., Conclusions: 3D molecular ultrasound using BR55 is very well suited to depicting the angiogenic activity in very small breast lesions, suggesting its potential for detecting and characterising these lesions.

Objective: The aim of the present study was to evaluate different statements in the literature on the influence of the sampling site on various blood parameters in a larger amount of animals. In addition, the practical usability of blood sampling from the udder vein or other accessible veins (e. g. saphenous vein or tail vein) in comparison to the jugular vein for metabolic analyses should be verified., Material and Methods: For this purpose blood samples from 92 cows and heifers were taken from the jugular vein, the udder vein and the saphenous vein of the left and right hind limb at four different time points (from 3 weeks ante partum until 8 weeks post partum) and comparatively investigated., Results: For five of the 16 investigated parameters (free fatty acids, beta-hydroxybutyrate, glucose, creatine kinase, and calcium) partially high significant differences between the sampling sites became apparent, dependant on the moment of analysis., Conclusion and Clinical Relevance: In metabolic analyses, which comprise the aforementioned parameters, the blood sampling site has decisively influence and has to be considered in the interpretation. The udder vein represents no alternative for blood sampling in high yielding cows for metabolic analyses. For obtaining meaningful results, the jugular vein has to be used for blood sampling, despite the higher procedural effort. An exception is the determination of the creatine kinase activity. Since defence movements of the animal's neck seem to have an influence on the local activity, the udder vein should be preferred as sampling site.

Studies show that elevated insulin-like growth factor-1 (IGF-1) levels are associated with an increased risk of breast cancer; however, mechanisms through which IGF-1 promotes mammary tumorigenesis in vivo have not been fully elucidated. To assess the possible involvement of COX-2 signaling in the pro-tumorigenic effects of IGF-1 in mammary glands, we used the unique BK5.IGF-1 mouse model in which transgenic (Tg) mice have significantly increased incidence of spontaneous and DMBA-induced mammary cancer compared to wild type (WT) littermates. Studies revealed that COX-2 expression was significantly increased in Tg mammary glands and tumors, compared to age-matched WTs. Consistent with this, PGE(2) levels were also increased in Tg mammary glands. Analysis of expression of the EP receptors that mediate the effects of PGE(2) showed that among the four G-protein-coupled receptors, EP3 expression was elevated in Tg glands. Up-regulation of the COX-2/PGE(2) /EP3 pathway was accompanied by increased expression of VEGF and a striking enhancement of angiogenesis in IGF-1 Tg mammary glands. Treatment with celecoxib, a selective COX-2 inhibitor, caused a 45% reduction in mammary PGE(2) levels, attenuated the influx of mast cells and reduced vascularization in Tg glands. These findings indicate that the COX-2/PGE(2) /EP3 signaling pathway is involved in IGF-1-stimulated mammary tumorigenesis and that COX-2-selective inhibitors may be useful in the prevention or treatment of breast cancer associated with elevated IGF-1 levels in humans. © 2011 Wiley Periodicals, Inc., (Copyright © 2011 Wiley Periodicals, Inc.)

This manuscript investigates, by Color Doppler ultrasonography, the basal hemodynamic features of the left cranial epigastric vein (milk vein) of 210 Holstein-Friesian cows, throughout lactation. Blood velocity, diameter, blood flow of the milk vein (before and after milking) and milk production detected in cows administered oxytocin were compared with the same parameters found in untreated cows. Blood velocity tended to decrease throughout lactation as did milk production, whereas both diameters and blood flows tended to increase towards the end of the study. All the four parameters showed higher values in the treated cows than in the untreated ones. This study provides a detailed picture of the hemodynamic features of the milk vein of the dairy cows throughout lactation and suggest that oxytocin may exert a vasodilatory effect, in vivo, at least on the cranial epigastric vein of the dairy cow., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

To test the hypothesis that reduction in dietary CP concentration coupled with crystalline AA inclusion increases the efficiency of AA use for milk production, mammary AA arteriovenous concentration differences (A-V), AA transport efficiency (A-V/A × 100), and transcript abundance of AA transporters and milk protein genes were determined in lactating sows fed 1 of 3 diets containing 9.5% (Deficient), 13.5% (Ideal), or 17.5% (Standard) CP, with a similar profile of indispensable and dispensable AA. On d 7 and 18, arterial and mammary venous blood and mammary tissue were sampled postfeeding. Transcript abundance of AA transporters b(0,+)AT (SLC7A9), y(+)LAT2 (SLC7A6), ATB(0,+) (SLC6A14), CAT-1 (SLC7A1), and CAT-2b (SLC7A2) and milk protein β-casein (CSN2) and LALBA (α-lactalbumin) were determined using reverse transcription quantitative PCR. Piglet ADG increased curvilinearly (linear and quadratic, P < 0.03) with increasing percent CP from Deficient to Standard. On d 7, Lys and Arg A-V and transport efficiency increased quadratically (P < 0.05) with increasing percent CP. On d 18, Lys A-V tended to increase (linear, P = 0.08) with increasing percent CP. Increasing CP increased Ile and Val A-V on d 7 (linear, P = 0.05 and P = 0.08, respectively) and Leu and Val on d 18 (linear, P = 0.07 and P = 0.04, respectively). On d 7, plasma concentrations of branched chain AA (BCAA):Lys decreased quadratically (P < 0.05). Expression of genes SLC7A9, SLC7A6, SLC6A14, SLC7A1, SLC7A2, CSN2, and LALBA was unaffected by diet. In conclusion, decreasing the dietary CP from 17.5% to 13.5% with inclusion of crystalline AA did not affect piglet ADG, AA transporter, or milk protein gene expression but increased mammary transport efficiency and A-V of Lys and Arg on d 7 of lactation. This increase was associated with a decrease in plasma concentration of BCAA:Lys, suggesting a competitive mechanism between cationic and BCAA for transport of AA across mammary cells.

Background: This study investigated the effect of milk yield on blood flow variables in the milk vein and musculophrenic vein in dairy cows., Methods: Five healthy dry cows, five cows with a daily milk yield of 10 kg and five others with a daily milk yield of 20 kg underwent B-mode and colour Doppler sonographic examination. The diameter of the veins, blood flow velocities and blood flow volumes were measured on both sides in standing, non-sedated cows using a 7.5 MHz linear transducer., Results: Lactating cows had significantly higher blood flow velocities in the milk vein than dry cows; the maximum blood flow velocity of dry cows and those with a daily milk yield of 10 and 20 kg were 14.04, 38.77 and 39.49 cm/s, respectively, the minimum velocities were 0.63, 3.02 and 2.64 cm/s, respectively, and the mean maximum velocities were 8.21, 26.67 und 28.22 cm/s, respectively. Cows producing 20 kg of milk a day had a blood flow volume of 3.09 l/min, which was significantly higher than 0.79 l/min recorded in dry cows. Lactating cows had significantly higher mean maximum blood flow velocities in the musculophrenic vein than dry cows. Blood flow variables of both veins did not differ significantly between the left and right side., Conclusion: This study showed that milk yield has a profound effect on blood flow variables in the milk vein and to a lesser extent the musculophrenic vein. This must be taken into consideration in future Doppler sonographic studies of these veins and possibly other vessels. Furthermore, measurements on one side are representative of both sides.

Tumor formation is an extensive process requiring complex interactions that involve both tumor cell-intrinsic pathways and soluble mediators within the microenvironment. Tumor cells exploit the intrinsic functions of many soluble molecules, including chemokines and their receptors, to regulate pro-tumorigenic phenotypes that are required for growth and progression of the primary tumor. Previous studies have shown that activation of inducible FGFR1 (iFGFR1) in mammary epithelial cells resulted in increased proliferation, migration, and invasion in vitro and tumor formation in vivo. These studies also demonstrated that iFGFR1 activation stimulated recruitment of macrophages to the epithelium where macrophages contributed to iFGFR1-mediated epithelial cell proliferation and angiogenesis. The studies presented here further utilize this model to identify the mechanisms that regulate FGFR1-induced macrophage recruitment. Results from this study elucidate a novel role for the inflammatory chemokine CX3CL1 in FGFR1-induced macrophage migration. Specifically, we illustrate that activation of both the inducible FGFR1 construct in mouse mammary epithelial cells and endogenous FGFR in the triple negative breast cancer cell line, HS578T, leads to expression of the chemokine CX3CL1. Furthermore, we demonstrate that FGFR-induced CX3CL1 is sufficient to recruit CX3CR1-expressing macrophages in vitro. Finally, blocking CX3CR1 in vivo leads to decreased iFGFR1-induced macrophage recruitment, which correlates with decreased angiogenesis. While CX3CL1 is a known target of FGF signaling in the wound healing environment, these studies demonstrate that FGFR activation also leads to induction of CX3CL1 in a tumor setting. Furthermore, these results define a novel role for CX3CL1 in promoting macrophage recruitment during mammary tumor formation, suggesting that the CX3CL1/CX3CR1 axis may represent a potential therapeutic approach for targeting breast cancers associated with high levels of tumor-associated macrophages.

Little is known about modifications of the mammary utilization of nutrients circulating in blood plasma when milk yield is strongly decreased by once-daily milking. A trial was carried out to describe the mammary nutritional adjustments linked to the downregulation of milk synthesis as milk accumulated over an extended milking interval in the bovine udder. Three Holstein dairy cows yielding 34.0 kg/d of milk were fitted with an ultrasound flow probe around the left external pudic artery and with catheters inserted into the left carotid and milk vein to estimate mammary blood flow (MBF) and mammary uptake of acetate, β-hydroxybutyrate, nonesterified fatty acids, glycerol, glucose, O(2), and CO(2) release. The trial was carried out over 2 consecutive weeks, with wk 2 repeating wk 1. Cows were milked twice daily at 12-h milking intervals. On d 3, cows were milked at 0630 h and were not milked for 36 h until d 4 at 1830 h. Over the following days, twice-daily milking was resumed using 12-h milking intervals. Each half-udder was milked separately. Secretion rates of milk and milk proteins decreased 67% during the 12-to-36-h interval of milk accumulation, whereas that of milk fat fell 30%. Timing of changes in MBF and lactose levels in blood plasma was concomitant and significant after 19.5 and 21.5h of milk accumulation in the udder, respectively. The MBF decreased, most likely because the usual increases in MBF no longer occurred when the udder was full of milk. After 24h of milk accumulation, MBF did not increase further when cows lay down, and did not increase as usual 3h after a meal, suggesting a possible physical effect of milk accumulated in the udder on MBF, complementing metabolic regulation. Mammary uptake or release of nutrients was lowered before 24h for glucose, acetate, and β-hydroxybutyrate and after 24h for total glycerol, O(2), and CO(2), mostly associated with the impaired MBF. However, these decreases ranged from 12 to 17%, and cannot entirely explain the -45 and -20% decreases in milk secretion rates observed during the entire 36 h of milk accumulation, thus confirming the primary role of intramammary metabolic regulation in the downregulation of milk secretion. The larger amount of nutrients taken up by the udder could explain the enhanced milk fat levels, involving a strongly modified metabolic fate of nutrients., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Prenatal exposure to BPA disturbs mammary gland histoarchitecture and increases the carcinogenic susceptibility to chemical challenges administered long after BPA exposure. Our aim was to assess the effect of prenatal BPA exposure on mammary gland angiogenesis and steroid hormone pathways in virgin cycling rats. Pregnant Wistar rats were exposed to either 25 or 250 g/kg/day (25 and 250 BPA, respectively) or to vehicle. Female offspring were autopsied on postnatal day (PND) 50 or 110. Ovarian steroid serum levels, the expression of steroid receptors and their co-regulators SRC-3 and SMRT in the mammary gland, and angiogenesis were evaluated. At PND 50, all BPA-treated animals had lower serum levels of progesterone, while estradiol levels remained unchanged. The higher dose of BPA increased mammary ERα and decreased SRC-3 expression at PND 50 and PND 110. SMRT protein levels were similar among groups at PND 50, whereas at PND 110, animals exposed to 250 BPA showed a lower SMRT expression. Interestingly, in the control and 25 BPA groups, SMRT increased from PND 50 to PND 110. At PND 50, an increased vascular area associated with higher VEGF expression was observed in the 250 BPA-treated rats. At PND 110, the vascular area was still increased, but VEGF expression was similar to that of control rats. The present results demonstrate that prenatal exposure to BPA alters the endocrine environment of the mammary gland and its angiogenic process. Increased angiogenesis and altered steroid hormone signals could explain the higher frequency of pre-neoplastic lesions found later in life. This article is part of a Special Issue entitled 'Endocrine disruptors'., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

As with most implanted biomaterials, the wound healing response following implantation of a silicone breast implant leads to the formation of a fibrotic capsule. This can result in capsular contracture, a painful complication that often necessitates the removal of implant. It is well established that nicotine and nicotinic agonists inhibit inflammatory signaling. Based on the link between the inflammatory response and capsule formation, we hypothesized that local delivery of nicotine from the implant may lead to the reduction in inflammation and capsule thickness, which may ultimately reduce the incidence of capsular contracture. Nicotine was loaded into PDMS membranes using a previously established method. The loaded materials were implanted into the submammary pockets between the third and fourth mammary glands of rats. To confirm that the nicotine was acting locally and not systemically, serum cotinine, the primary metabolite of nicotine, was measured by ELISA at 3 days. Thirty days post implantation, the animals were euthanized and the tissue samples were fixed for histological analysis. Blood vessel density was measured immunohistochemically, while the capsule thickness was evaluated microscopically. While the presence of the nicotine metabolite, cotinine, in the serum at the early time points demonstrated that the nicotine was released locally from the devices, there were no significant differences in the capsule thickness between the control and experimental implants. However, the results indicated that there were differences in angiogenesis with the local delivery of nicotine, which may have other implications for the development of biomaterials.

Recent dynamic contrast-enhanced MRI studies using the adiabatic tissue homogeneity model have highlighted potential issues of difficulty in convergence during data-fitting and reduced parameter precision, due to discontinuities in the adiabatic tissue homogeneity model. This study presents two solutions (an analytic approach and a discrete correction method) to such convergence problems and show that these problems can be attributed to an inaccurate approximation of the convolution integral based on the standard trapezoidal quadrature. It is further explained that such issues of discontinuity in the impulse residue function do not pertain only to the adiabatic tissue homogeneity model, but are generic to all tracer kinetic models, if the difference in bolus arrival time between the arterial input and tissue voxels were to be accounted for simultaneously during model-fitting., (Copyright © 2011 Wiley-Liss, Inc.)

Objectives were to determine the effects of maternal dietary supranutritional Se and nutritional plane during gestation on capillary surface density, capillary area density, and angiogenic factor expression in the developing mammary gland of primiparous ewes. Selenium treatments were initiated at breeding [adequate Se (ASe; 9.5 μg/kg of body weight) vs. high Se (HSe; 81.8 μg/kg of body weight)] and nutritional planes at d 50 of gestation [Low, 60%; moderate (Mod), 100%; and High, 140% of requirements). Mammary glands were collected within 24h postpartum. Vascular development was assessed in the glandular portion of the mammary gland. Vascularity was determined for mammary tissue with the following measurements taken: the cross-sectional capillary area density (total capillary area as a proportion of tissue area) and capillary surface density (CSD; total capillary circumference per unit of tissue area). High-Se ewes had greater capillary surface and area densities compared with ASe ewes. A tendency existed for an Se × plane of nutrition interaction for CSD with maternal diet not affecting CSD in HSe ewes, but Low ewes had a decreased CSD compared with Mod ewes, with High being intermediate in ASe ewes. Moreover, HSe-Low and HSe-High ewes had increased CSD compared with ASe-Low and ASe-High, respectively. Although Se status did not influence angiogenic factor mRNA expression, mammary glands from Low ewes tended to have increased VEGF and FLT1 mRNA expression compared with High ewes, with Mod being intermediate. Maternal plane of nutrition did not affect mammary gland glutathione peroxidase activity, but it was increased in HSe compared with ASe ewes. Increased mammary capillary nutrient exchange area may contribute to previously observed changes in colostrum quality., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The objectives of the current study were to determine the fate and contribution to Met kinetics of 2-hydroxy-4-(methylthio)butanoate (HMTBA) at the whole-body, splanchnic, and mammary levels. Four multicatheterized cows (31.3 kg of milk/d; 17.7 kg of DMI/d) were used in a crossover design, with two 1-wk periods, to determine the metabolic fate of HMTBA and its effect on Met metabolism. Over the last 2 d of each period, cows were infused, via a jugular vein, with saline or HMTBA (Alimet, Novus International Inc., St. Louis, Mo) at the rate of 36 g/d. During the last 8h, the HMTBA infusion was substituted by equimolar [1-(13)C]HMTBA (8.79 mmol/h) and l[methyl-(2)H(3)]Met (1.31 mmol/h) was infused in all cows. During the last 5h, hourly samples (n=6) were collected to determine plasma flows plus the isotopic enrichments (IE) and concentrations of HMTBA ((13)C) and Met (both (13)C and (2)H(3)) in plasma from an artery plus portal, hepatic, and mammary veins. The IE of [(13)C] and [(2)H(3)]Met were also determined in milk protein taken over the last 1h of infusion in HMTBA-infused cows. The infused HMTBA increased whole-body plasma flux of Met by 6.5 mmol/h (from 17.9 to 24.4 mmol/h). Based on enrichments of (13)C-labeled Met, 3.8 mmol/h of Met flow through plasma was derived directly from HMTBA. These 2 estimates accounted for between 43 to 74% of the HMTBA dose infused, contributing to increased whole-body Met availability. Although the portal-drained viscera, liver, and mammary gland (MG) extracted 11, 37, and 3.4%, respectively, of the infused HMTBA, tissue net Met fluxes were either unchanged (portal-drained viscera, MG) or even reduced (liver: -7.9 vs. -2.4±0.6 mmol/h). Therefore, net postsplanchnic supply of Met decreased from 7.0 to 2.9 mmol/h between control and HMTBA-infused cows, compared with needs for milk protein secretion of 7.6 and 8.1 mmol/h, respectively. The HMTBA provided directly 15% of the Met required for milk protein secretion, with 0.2 mmol/h synthesized within the MG, whereas 1.1 mmol/h originated from Met produced in other tissues and transported to the MG through blood circulation. Most of the remainder needed by the MG arose from unlabeled Met released from protein breakdown in extra-splanchnic tissues and that was not reused to support intracellular protein synthesis, as this function was performed by Met synthesized from HMTBA in situ. Absorbed HMTBA, therefore, both produces and spares Met for use by the MG., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Fibronectin (Fn) plays an important part in the branching morphogenesis of salivary gland, lung, and kidney. Here, we examine the effect of the conditional knockout of Fn in the mammary epithelium [Fn(MEp-/-)] on postnatal mammary gland development, using Cre-loxP-mediated gene knockout technology. Our data show that Fn deletion causes a moderate retardation in outgrowth and branching of the ductal tree in 5-week-old mice. These defects are partially compensated in virgin 16-week-old mice. However, mammary glands consisting of Fn-deficient epithelial cells fail to undergo normal lobuloalveolar differentiation during pregnancy. The severity of lobuloalveolar impairment ranged from lobular hypoplasia to aplasia in some cases and was associated with the amount of Fn protein recovered from these glands. Decreased rates of mammary epithelial cell proliferation accounted for delayed ductal outgrowth in virgin and lack of alveologenesis in pregnant Fn(MEp-/-) mice. Concomitant decreased expression of integrin beta(1) (Itgb1) and lack of autophosphorylation of focal adhesion kinase (Fak) suggest that this pathology might, at least in part, be mediated by disruption of the Fn/Itgb1/Fak signaling pathway., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Concomitant with the extensive growth and differentiation of the mammary epithelium during pregnancy and lactation, and epithelial involution after weaning, the vasculature of the mammary gland undergoes repeated cycles of expansion and regression. Vascular expansion is effected by sprouting angiogenesis, intussusception and conceivably also vasculogenesis. The capacity of the epithelial cells to stimulate vascular growth and differentiation is dependent on the constellation of systemic and local hormones and growth factors as well as the changing demands for oxygenation and nutrient supply. This results in the release of angiogenic factors which stimulate endothelial cell growth and regulate vascular architecture. In contrast to the angiogenic phase of the mammary gland cycle, little is known about the control of vascular regression although this would possibly offer new insights into therapeutic possibilities against breast cancer. In this review we summarize knowledge regarding the mechanisms regulating the vasculature of the mammary gland and delineate the importance of the vasculature in the attainment of organ function. In addition, we discuss the angiogenic mechanisms observed during mammary carcinogenesis and their consequences for breast cancer therapy.

During the normal course of lactation, mammary metabolic activity and blood flow are closely correlated. Six lactating goats were used in this experiment to test the hypothesis that the capillary network and the capillary enzyme, carbonic anhydrase (CA; EC 4.2.1.1) are important regulatory factors involved in the coordination of mammary blood flow (MBF) and metabolic activity. Milk vein blood velocity was determined as a measure of MBF, and fine needle mammary biopsies were obtained at different time points during lactation and by the end of a 14-d bovine somatotropin (BST) treatment initiated 3 months post partum. In mammary sections, CA activity was determined histochemically and alveolar and capillary structures by image analyses upon azure blue staining. In early lactation, alveoli were large and surrounded by many small capillaries with high CA activity. As lactation progressed, capillaries almost tripled in size, whereas number of capillaries surrounding each alveolus decreased by 1/3, and CA activity more than halved. BST treatment did not affect capillary traits but increased number of alveoli in mammary sections, and BST thus appeared to be targeted mostly towards the mammary epithelial cell. Milk vein blood velocity decreased over the course of lactation, when capillary area markedly increased, suggesting that control of mammary blood perfusion is not at the level of the capillary itself, but at pre- or post-capillary sites. We suggest that the observed changes in capillary diameter and CA activity with progressing lactation contributes to reduce efficiency of nutrient and waste product exchange across the capillary-mammary epithelial cell barrier, and this could be an important factor in regulation of mammary (epithelial cell) metabolic activity and lactation performance.

Recently, we found that mutation of the C-terminus of transcription factor hexamethylene bisacetamide-inducible protein 1 (HEXIM1) in mice leads to abnormalities in cardiovascular development because of aberrant vascular endothelial growth factor (VEGF) expression. HEXIM1 regulation of some genes has also been shown to be positive transcription elongation factor b (P-TEFb) dependent. However, it is not known whether HEXIM1 regulates VEGF in the mammary gland. We demonstrate that HEXIM1 regulates estrogen-induced VEGF transcription through inhibition of estrogen receptor-alpha recruitment to the VEGF promoter in a P-TEFb-independent manner in MCF-7 cells. Under hypoxic conditions, HEXIM1 inhibits estrogen-induced hypoxia-inducible factor-1 alpha (HIF-1alpha) protein expression and recruitment of HIF-1alpha to the hypoxia-response element in the VEGF promoter. In the mouse mammary gland, increased HEXIM1 expression decreased estrogen-driven VEGF and HIF-1alpha expression. Conversely, a mutation in the C-terminus of HEXIM1 (HEXIM1(1-312)) led to increased VEGF and HIF-1alpha expression and vascularization in mammary glands of heterozygous HEXIM1(1-312) mice when compared with their wild-type littermates. In addition, HEXIM1(1-312) mice have a higher incidence of carcinogen-induced mammary tumors with increased vascularization, suggesting an inhibitory role for HEXIM1 during angiogenesis. Taken together, our data provide evidence to suggest a novel role for HEXIM1 in cancer progression.