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The link between in utero and early life insults and the development of chronic illness remains to be fully understood, but there is increasing data to indicate that microvasculature pathology plays an important mechanistic role. Currently available data indicate that retinal microvasculature changes are detectable in children as young as six years of age, however, there are no data for younger children. We present retinal microvasculature measurement from the first two years of life. Retinal images suitable for analysis were available from 18 infants in our proof-of-concept study. The mean and standard deviation (SD) for birth weight and gestation was 3410 (384) g and 39.1(1.4) weeks, respectively. Retinal vessel calibres were summarized as the mean(SD) central retinal arteriolar equivalent (CRAE) at six months of age was 156 (32) μm, increased to 175 (75) μm by 12 months and a slightly declined by 24 months of age to 168 (50) μm. In a similar pattern, mean(SD) central retinal venular equivalent (CRVE) at six months was 211 (19) μm, increased to 238 (25) μm by 12 months of age followed by a slight decline at 24 months of age to 222 (36) μm. The arterio-venous ratio and tortuosity index remained the same at 6, 12 and 24 months. Findings from this study could help future investigators better understand early microvasculature changes and adaptation that occur early in life., (Copyright © 2019 Elsevier Inc. All rights reserved.)

It is unknown whether aging or exercise training affect the dynamics of arteriolar vasoconstriction., Purpose: We hypothesized that old age will slow, and exercise training will speed, the dynamics of skeletal muscle arteriolar vasoconstriction in resistance vessels of aged rats., Method: Young (6 month old) and aged (24 month old) male Fischer-344 rats were assigned to sedentary (Sed: n = 6/age group) or exercise-trained (ET: n = 5 aged and 6 young; via treadmill running for 10-12 weeks) groups. After completion of training, arterioles from the red portion of the gastrocnemius muscle were removed, cannulated, and exposed to 10 -4  M norepinephrine (NE) or 20 mM caffeine. Changes in luminal diameter were recorded for analysis of constrictor dynamics., Result: Old age blunted all kinetic parameters (i.e., time delay, time constant) resulting in vasoconstriction taking ~3 times as long to reach a steady state (SS) versus younger counterparts for NE (aged-sed: 15.6 ± 6.0 versus young-sed: 4.6 ± 0.5 s; P < 0.05) with a similar time course to caffeine. Exercise training resulted in a similar time to SS between age groups for NE (aged-ET: 6.8 ± 1.6 versus young-ET: 7.0 ± 0.6 s) and caffeine (aged-ET: 7.8 ± 0.6 versus young-ET: 8.6 ± 1.0 s)., Conclusion: The results of this study demonstrate that aging blunts the rate of vasoconstriction in skeletal muscle resistance vessels to the sympathetic neurotransmitter NE due, in part, to an attenuated rate of contraction from intracellular calcium release. Further, exercise training speeds the dynamics of constriction to both NE and caffeine with old age.

Previous studies have documented that rapid juvenile growth is accompanied by functional changes in the arteriolar endothelium, but much less is known about functional changes in arteriolar smooth muscle over this period. In this study, we investigate the possible contribution of epithelial sodium channels (ENaC) to the myogenic behaviour of arterioles at two stages of juvenile growth. The effects of the ENaC inhibitor benzamil on different levels of myogenic tone were studied in isolated gracilis muscle arterioles from rats aged 21-28 days ("weanlings") and 42-49 days ("juveniles"). ENaC subunit expression in the arteriolar wall was also determined, and the interaction between ENaC and nitric oxide (NO) in regulating vascular tone was explored by combined use of benzamil and N G -monomethyl-l-arginine (l-NMMA). At physiological pressures, both steady-state myogenic tone and the dynamic adjustments in this tone triggered by acute pressure changes were less in juvenile arterioles than in weanling arterioles. α, β and γ ENaC protein was present in arterioles at both ages, but benzamil only had an effect on myogenic tone in weanling arterioles. In these vessels, benzamil increased, rather than decreased, myogenic tone, and this effect was prevented by l-NMMA or endothelial removal. These findings suggest that although ENaC is present in gracilis muscle arterioles of both weanling and juvenile rats, it is not obligatory for the genesis of myogenic activity in these vessels at either age. However, ENaC activity can significantly modulate the level of myogenic tone through stimulation of endothelial NO release at an early stage of growth., (© 2016 John Wiley & Sons Australia, Ltd.)

Tissue repair processes are characterized by the biphasic recruitment of distinct subpopulations of blood monocytes, including classical ("inflammatory") monocytes (IMs, Ly6C(hi)Gr1(+)CX3CR1(lo)) and non-classical anti-inflammatory monocytes (AMs, Ly6C(lo)Gr1(-)CX3CR1(hi)). Drug-eluting biomaterial implants can be used to tune the endogenous repair process by the preferential recruitment of pro-regenerative cells. To enhance recruitment of AMs during inflammatory injury, a novel N-desulfated heparin-containing poly(ethylene glycol) diacrylate (PEG-DA) hydrogel was engineered to deliver exogenous stromal derived factor-1α (SDF-1α), utilizing the natural capacity of heparin to sequester and release growth factors. SDF-1α released from the hydrogels maintained its bioactivity and stimulated chemotaxis of bone marrow cells in vitro. Intravital microscopy and flow cytometry demonstrated that SDF-1α hydrogels implanted in a murine dorsal skinfold window chamber promoted spatially-localized recruitment of AMs relative to unloaded internal control hydrogels. SDF-1α delivery stimulated arteriolar remodeling that was correlated with AM enrichment in the injury niche. SDF-1α, but not unloaded control hydrogels, supported sustained arteriogenesis and microvascular network growth through 7 days. The recruitment of AMs correlated with parameters of vascular remodeling suggesting that tuning the innate immune response by biomaterial SDF-1α release is a promising strategy for promoting vascular remodeling in a spatially controlled manner., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aims: It is still controversial whether bone marrow (BM)-derived endothelial progenitor cells (EPCs) can contribute to vascular repair and prevent the progression of vascular diseases. We aimed to characterize BM-derived EPC subpopulations and to evaluate their therapeutic efficacies to repair injured vascular endothelium of systemic and pulmonary arteries., Methods and Results: BM mononuclear cells of Fisher-344 rats were cultured under endothelial cell-conditions. Early EPCs appeared on days 3-6. Late-outgrowth and very late-outgrowth EPCs (LOCs and VLOCs) were defined as cells forming cobblestone colonies on days 9-14 and 17-21, respectively. Among EPC subpopulations, LOCs showed the highest angiogenic capability with enhanced proliferation potential and secretion of proangiogenic proteins. To investigate the therapeutic effects of these EPCs, Fisher-344 rats underwent wire-mediated endovascular injury in femoral artery (FA) and were concurrently injected intraperitoneally with 60mg/kg monocrotaline (MCT). Injured rats were then treated with six injections of one of three EPCs (1×10(6) per time). After 4weeks, transplanted LOCs, but not early EPCs or VLOCs, significantly attenuated neointimal lesion formation in injured FAs. Some of CD31(+) LOCs directly replaced the injured FA endothelium (replacement ratio: 11.7±7.0%). In contrast, any EPC treatment could neither replace MCT-injured endothelium of pulmonary arterioles nor prevent the progression of pulmonary arterial hypertension (PAH). LOCs modified protectively the expression profile of angiogenic and inflammatory genes in injured FAs, but not in MCT-injured lungs., Conclusion: BM-derived LOCs can contribute to vascular repair of injured systemic artery; however, even they cannot rescue injured pulmonary vasculature under MCT-induced PAH., (Copyright © 2015. Published by Elsevier Ltd.)

Arteriogenesis-the growth of collateral arterioles-partially compensates for the progressive occlusion of large conductance arteries as it may occur as a consequence of coronary, cerebral or peripheral artery disease. Despite being clinically highly relevant, mechanisms driving this process remain elusive. In this context, our study revealed that abundance of regulator of G-protein signalling 5 (RGS5) is increased in vascular smooth muscle cells (SMCs) of remodelling collateral arterioles. RGS5 terminates G-protein-coupled signalling cascades which control contractile responses of SMCs. Consequently, overexpression of RGS5 blunted Gαq/11-mediated mobilization of intracellular calcium, thereby facilitating Gα12/13-mediated RhoA signalling which is crucial for arteriogenesis. Knockdown of RGS5 evoked opposite effects and thus strongly impaired collateral growth as evidenced by a blockade of RhoA activation, SMC proliferation and the inability of these cells to acquire an activated phenotype in RGS5-deficient mice after the onset of arteriogenesis. Collectively, these findings establish RGS5 as a novel determinant of arteriogenesis which shifts G-protein signalling from Gαq/11-mediated calcium-dependent contraction towards Gα12/13-mediated Rho kinase-dependent SMC activation., (© 2014 The Authors. Published under the terms of the CC BY 4.0 license.)

Objectives: We hypothesized that trajectories of adiposity across childhood would be associated with retinal microcirculatory diameters at age 12 years, independent of BP., Methods: The ALSPAC followed a cohort of children born in 1991-1992. The current study includes all children with retinal images acquired at the 12 years clinic and individual trajectories of PI from 0 to 2 years and BMI from 2 to 10 years. Retinal microvascular measures included retinal arteriolar and venular diameters., Results: Children in this analysis had a birth weight of 3.5 ± 0.4 kg, a PI of 26.2 ± 2.4 kg/m(3) and a gestational age of 39.7 ± 1.4 weeks (mean ± SD). Analysis of growth trajectories showed that lower PI at birth was associated with narrower retinal arterioles. Higher PI at birth was associated with wider venular diameter, and a stronger positive association was evident between BMI change at 5-5.5 and 8.5-10 years with wider venular diameters. Current fat mass was also associated with wider venular diameters., Conclusions: Retinal arterioles and venules are differentially associated with growth in early life and childhood adiposity. Early adiposity may adversely affect the microcirculation, with important implications for cardiovascular risk in adulthood., (© 2013 John Wiley & Sons Ltd.)

Collateral arterioles enlarge in both diameter and length, and develop corkscrew-like tortuous patterns during remodeling. Recent studies showed that artery buckling could lead to tortuosity. The objective of this study was to determine arteriole critical buckling pressure and buckling pattern during arteriole remodeling. Arterioles were modeled as elastic cylindrical vessels with an elastic matrix support and underwent axial and radial growth. Our results demonstrated that arteriole critical buckling pressure decreased with increasing axial growth ratio and radius growth ratio, but increased with increasing wall thickness. Arteriole buckling mode number increased (wavelength decreased) with increasing axial growth ratio, but decreased with increasing radius growth ratio and wall thickness. Our study suggests that axial growth in arterioles makes them prone to buckling and that buckling leads to tortuous collaterals. These results shed light on the mechanism of collateral arteriole tortuosity., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: Insufficient angiogenesis and arteriogenesis in cardiac tissue after myocardial infarction (MI) is a significant factor hampering the functional recovery of the heart. To overcome this problem we screened for compounds capable of stimulating angiogenesis, and herein investigate the most active molecule, 5-Methoxyleoligin (5ML), in detail., Methods and Results: 5ML potently stimulated endothelial tube formation, angiogenic sprouting, and angiogenesis in a chicken chorioallantoic membrane assay. Further, microarray- and knock down- based analyses revealed that 5ML induces angiogenesis by upregulation of CYP26B1. In an in vivo rat MI model 5ML potently increased the number of arterioles in the peri-infarction and infarction area, reduced myocardial muscle loss, and led to a significant increase in LV function (plus 21% 28 days after MI)., Conclusion: The present study shows that 5ML induces CYP26B1-dependent angiogenesis in vitro, and arteriogenesis in vivo. Whether or not CYP26B1 is relevant for in vivo arteriogenesis is not clear at the moment. Importantly, 5ML-induced arteriogenesis in vivo makes the compound even more interesting for a post MI therapy. 5ML may constitute the first low molecular weight compound leading to an improvement of myocardial function after MI.

Male rat renal blood vessels undergo reduced contraction to norepinephrine with aging. There is a greater renal vascular impairment in male compared with female rats. We investigated specific tyrosine kinase receptor inhibition of renal interlobar artery responsiveness to phenylephrine in male and female rats at specifically designated ages. Vessels from young male rats contracted much less to phenylephrine when the vessels were pretreated with the tyrosine kinase inhibitors Lavendustin A, HNMPA-(AM)₃, or AG1478. Vessels from adult female rats pretreated with Lavendustin A showed no difference in contraction from control, but did demonstrate a slightly reduced contraction when pretreated with AG1478. Middle-aged male rat vessels treated with Lavendustin A demonstrated no inhibition, but the insulin and epidermal growth factor receptor (EGFR) antagonists both induced a decline in contraction. Vessels from aged male rats demonstrated no effect related to the 3 pretreatments. Middle-aged and aged female rats pretreated with any inhibitor demonstrated no inhibitor-dependent alterations. We conclude that maximum contraction of interlobar arteries from adult male rats is reduced when tyrosine kinase receptor activity is reduced. Female rats demonstrated much less inhibitor-related change of contraction.

Nitric oxide (NO) mediates a major portion of arteriolar endothelium-dependent dilation in adults, but indirect evidence has suggested that NO contributes minimally to these responses in the young. Isolated segments of arterioles were studied in vitro to verify this age-related increase in NO release and investigate the mechanism by which it occurs. Directly measured NO release induced by ACh or the Ca(2+) ionophore A-23187 was five- to sixfold higher in gracilis muscle arterioles from 42- to 46-day-old (juvenile) rats than in those from 25- to 28-day-old (weanling) rats. There were no differences between groups in arteriolar endothelial NO synthase (eNOS) expression or tetrahydrobiopterin levels, and arteriolar l-arginine levels were lower in juvenile vessels than in weanling vessels (104 ± 6 vs.126 ± 3 pmol/mg). In contrast, agonist-induced eNOS Thr(495) dephosphorylation and eNOS Ser(1177) phosphorylation (events required for maximal activity) were up to 30% and 65% greater, respectively, in juvenile vessels. Juvenile vessels did not show increased expression of enzymes that mediate these events [protein phosphatases 1 and 2A and PKA and PKB (Akt)] or heat shock protein 90, which facilitates Ser(1177) phosphorylation. However, agonist-induced colocalization of heat shock protein 90 with eNOS was 34-66% greater in juvenile vessels than in weanling vessels, and abolition of this difference with geldanamycin also abolished the difference in Ser(1177) phosphorylation between groups. These findings suggest that growth-related increases in arteriolar NO bioavailability may be due at least partially to changes in the regulation of eNOS phosphorylation and increased signaling activity, with no change in the abundance of eNOS signaling proteins.

Microvessels live 'a life under pressure' in several ways. In a literal sense, vessels of the microcirculation are exposed to high levels of stress caused primarily by the intravascular pressure head. In a figurative sense, the individual vessel and the microvascular network as a whole must continuously strive to meet the changing demands of the surrounding tissue. The 'principle of optimal operation' as formulated by Y. C. Fung states that living tissues adapts structurally through remodelling and growth until a level of tensile and compressive stresses is reached at which tissue performance is optimal. This behaviour is characteristic for the microvascular wall. It is highly plastic by nature and meets sustained changes by structural adaptation so as to maintain functional optimality. Owing to the orientation of the vascular smooth muscle cell in the media, in particular, the circumferential stress component has a huge impact on the state of the vascular wall. It is involved as a unifying factor on vastly different timescales in processes as diverse as acute regulation of vessel diameter, structural vessel remodelling and growth or atrophy of the vascular wall. The aim of this MiniReview was to outline in brief this integrative role of circumferential wall stress in the microcirculation., (© 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society.)

The kidney is a highly vascularized organ that normally receives a fifth of the cardiac output. The unique spatial arrangement of the kidney vasculature with each nephron is crucial for the regulation of renal blood flow, GFR, urine concentration, and other specialized kidney functions. Thus, the proper and timely assembly of kidney vessels with their respective nephrons is a crucial morphogenetic event leading to the formation of a functioning kidney necessary for independent extrauterine life. Mechanisms that govern the development of the kidney vasculature are poorly understood. In this review, we discuss the anatomical development, embryological origin, lineage relationships, and key regulators of the kidney arterioles and postglomerular circulation. Because renal disease is associated with deterioration of the kidney microvasculature and/or the reenactment of embryonic pathways, understanding the morphogenetic events and processes that maintain the renal vasculature may open new avenues for the preservation of renal structure and function and prevent the progression of renal disease.

Objective: In the current study, we aimed to examine the associations of low birth weight with retinal vascular caliber and vascular fractal dimension during early adolescence., Methods: A population-based study of 12-year-old schoolchildren (2353/3144 [75.3%]) recruited from a random cluster sample of 21 schools. Birth weight, birth length and head circumference were obtained via parent report of the child's birth record. Retinal images were taken and vessel diameter and fractal dimension were quantified using validated computer-based methods., Results: After adjusting for age, sex, ethnicity, body mass index, iris color, axial length, mean arterial blood pressure, prematurity and fellow retinal vascular caliber, children in the lowest quartiles of birth weight had ∼2.5 μm narrower mean retinal arteriolar caliber than those in the highest quartiles (p for trend = 0.001). Associations were observed between shorter birth length and smaller head circumference with narrower retinal arterioles. Smaller head circumference was associated with decreased fractal dimension (p for trend = 0.03)., Conclusions: Children with lower birth weight were more likely to have narrower retinal arterioles, while those with smaller head circumference were more likely to have reduced complexity of their retinal microvasculature. These variations in microvascular structure in adolescence could reflect a susceptibility to cardiovascular disease during adulthood, resulting from a disadvantaged growth environment in utero., (© 2010 John Wiley & Sons Ltd.)

Objective: Chronic and acute ischemic diseases-peripheral artery disease, coronary artery disease, stroke-result in tissue damage unless blood flow is maintained or restored in a timely manner. Mice of different strains recover from arteriolar ligation (by increasing collateral blood flow) at different speeds. We quantify the spatio-temporal patterns of microvascular network remodeling following arteriolar ligation in different mouse strains to better understand inter-individual variability., Methods: Whole-muscle spinotrapezius microvascular networks of mouse strains C57Bl/6, Balb/c and CD1 were imaged using confocal microscopy following ligation of feeding arterioles., Results: Baseline arteriolar structures of C57Bl/6 and Balb/c mice feature heavily ramified arcades and unconnected dendritic trees, respectively. This network angioarchitecture identifies ischemia-protected and ischemia-vulnerable tissues; unlike C57Bl/6, downstream capillary perfusion in Balb/c spinotrapezius is lost following ligation. Perfusion recovery requires arterialization (expansion and investment of mural cells) of a subset of capillaries forming a new low-resistance collateral pathway between arteriolar trees. Outbred CD1 exhibit either Balb/c-like or C57Bl/6-like spinotrapezius angioarchitecture, predictive of response to arteriolar ligation., Conclusions: This collateral capillary arterialization process may explain the reported longer time required for blood flow recovery in Balb/c hindlimb ischemia, as low-resistance blood flow pathways along capillary conduits must be formed ("arterialization") before reperfusion.

Transplanting hematopoietic and peripheral blood-derived stem/progenitor cells can have beneficial effects in slowing the effects of heart failure. We investigated whether human bone marrow CD133(+)-derived cells (BM-CD133(+) cells) might be used for cell therapy of heart injury in combination with tissue engineering. We examined these cells for: 1) their in vitro capacity to be converted into cardiomyocytes (CMs), and 2) their potential for in vivo differentiation when delivered to a tissue-engineered type I collagen patch placed on injured hearts (group II). To ensure a microvascular network ready for use by the transplanted cells, cardiac injury and patching were scheduled 2 weeks before cell injection. The cardiovascular potential of the BM-CD133(+) cells was compared with that of a direct injection (group I) of the same cells in heart tissue damaged according to the same schedule as for group II. While a small fraction (2 ± 0.5%) of BM-CD133(+)cells cocultured with rat CMs switched in vitro to a CM-like cell phenotype, in vivo-and in both groups of nude rats transplanted with BM-CD133(+)--there was no evidence of any CM differentiation (as detected by cardiac troponin I expression), but there were signs instead of new capillaries and small arterioles. While capillaries prevailed over arterioles in group II, the opposite occurred in group I. The transplanted cells further contributed to the formation of new microvessels induced by the patch (group II) but the number of vessels did not appear superior to the one developed after directly injecting the BM-CD133(+)cells into the injured heart. Although chimeric human-rat microvessels were consistently found in the hearts of both groups I and II, they represented a minority (1.5-2.3%) compared with those of rat origin. Smooth muscle myosin isoform expression suggested that the arterioles achieved complete differentiation irrespective of the presence or absence of the collagen patch. These findings suggest that: 1) BM-CD133(+) cells display a limited propensity for in vitro conversion to CMs; 2) the preliminarily vascularized bioscaffold did not confer a selective homing and differentiation advantage for the phenotypic conversion of BM-CD133(+) cells into CMs; and 3) combined patching and cell transplantation is suitable for angiogenesis and arteriogenesis, but it does not produce better results, in terms of endothelial and smooth muscle cell differentiation, than the "traditional" method of cell injection into the myocardium.

In childhood, widening of Virchow-Robin spaces is rarely secondary to specific progressive disorders, but more often appears in poorly characterized developmental conditions. From data collected in a neuropediatric department, we examined whether clinical data associated with "constitutional widening of Virchow-Robin spaces" allowed delineation of recognizable entities. Signs in 10 patients, mostly boys, suggested nonspecific cerebral dysfunctions, e.g., developmental delay, nonspecific epilepsy, headaches, or benign macrocephaly. Spaces were sometimes round, subsequently mimicking microcystic malacic lesions. In two patients, abnormal magnetic resonance imaging signals were evident in white matter contiguous to widened perivascular spaces, suggesting a broader disorder of fluid exchanges. Four cases occurred in two sibships. In two families, other patients exhibited early developmental difficulties. Long-term clinical and magnetic resonance imaging surveillance will clarify which cases of primary Virchow-Robin space dilatation imply a benign prognosis. Performance of magnetic resonance imaging on any relative exhibiting minor neuropsychologic handicaps would permit estimations of real genetic incidence.

The response to hemodynamic force is implicated in a number of pathologies including collateral vessel development. However, the transcriptional effect of hemodynamic force is extremely challenging to examine in vivo in mammals without also detecting confounding processes such as hypoxia and ischemia. We therefore serially examined the transcriptional effect of preventing cardiac contraction in zebrafish embryos which can be deprived of circulation without experiencing hypoxia since they obtain sufficient oxygenation by diffusion. Morpholino antisense knock-down of cardiac troponin T2 (tnnt2) prevented cardiac contraction without affecting vascular development. Gene expression in whole embryo RNA from tnnt2 or control morphants at 36, 48, and 60 h postfertilization (hpf) was assessed using Affymetrix GeneChip Zebrafish Genome Arrays (>14,900 transcripts). We identified 308 differentially expressed genes between tnnt2 and control morphants. One such (CXCR4a) was significantly more highly expressed in tnnt2 morphants at 48 and 60 hpf than controls. In situ hybridization localized CXCR4a upregulation to endothelium of both tnnt2 morphants and gridlock mutants (which have an occluded aorta preventing distal blood flow). This upregulation appears to be of functional significance as either CXCR4a knock-down or pharmacologic inhibition impaired the ability of gridlock mutants to recover blood flow via collateral vessels. We conclude absence of hemodynamic force induces endothelial CXCR4a upregulation that promotes recovery of blood flow.

Background: Adipose tissue consists of mature adipocytes and a mononuclear cell fraction termed adipose tissue-derived cells (ADCs). Within these heterogeneous ADCs exists a mesenchymal stem cell-like cell population, termed adipose tissue-derived stem cells. An important clinical advantage of adipose tissue-derived stem cells over other mesenchymal stem cell populations is the fact that they can be isolated in real time in sufficient quantity, such that ex vivo expansion is not necessary to obtain clinically relevant numbers for various therapeutic applications., Materials and Methods: The aim of this investigation was to evaluate the therapeutic potential of freshly isolated ADCs in treating rats acutely following myocardial infarction. Rats underwent 45 min of left anterior descending artery occlusion followed by reperfusion. Fifteen minutes post-myocardial infarction, saline or 5 x 10(6) ADCs from green fluorescent protein-expressing transgenic rats were injected into the chamber of the left ventricle. Left ventricular function and morphometry was followed with 2-D echocardiography for 12 wk, at which point hearts were harvested for histological analysis., Results: Twelve weeks following cell therapy, left ventricular end-diastolic dimension was less dilated while the ejection fraction and cardiac output of ADC-treated rats were significantly improved compared to control rats (P < 0.01). Despite this benefit, absolute engraftment rates were low. This paradox may be partially explained by ADC-induced increases in both capillary and arteriole densities., Conclusions: These data confirm the therapeutic benefit of freshly isolated ADCs delivered post-MI and suggest a novel beneficial mechanism for ADCs through a potent proangiogenic effect.

Chemokine (C-C motif) receptor-2 (CCR2) regulates arteriogenesis and angiogenesis, facilitating the MCP-1-dependent recruitment of growth factor-secreting bone marrow-derived cells (BMCs). Here, we tested the hypothesis that the BMC-specific expression of CCR2 is also required for new arteriole formation via capillary arterialization. Following non-ischemic saphenous artery occlusion, we measured the following in gracilis muscles: monocyte chemotactic protein-1 (MCP-1) in wild-type (WT) C57Bl/6J mice by ELISA, and capillary arterialization in WT-WT and CCR2(-/-)-WT (donor-host) bone marrow chimeric mice, as well as BMC transdifferentiation in EGFP(+)-WT mice, by smooth muscle (SM) alpha-actin immunochemistry. MCP-1 levels were significantly elevated 1 day after occlusion in WT mice. In WT-WT mice at day 7, compared to sham controls, arterial occlusion induced a 34% increase in arteriole length density, a 46% increase in SM alpha-actin(+) vessels, and a 45% increase in the fraction of vessels coated with SM alpha-actin, indicating significant capillary arterialization. However, in CCR2(-/-)-WT mice, no differences were observed between arterial occlusion and sham surgery. In EGFP(+)-WT mice, EGFP and SM alpha-actin never colocalized. We conclude that BMC-specific CCR2 expression is required for skeletal muscle capillary arterialization following arterial occlusion; however, BMCs do not transdifferentiate into smooth muscle.

Cerebral, coronary, and peripheral artery diseases combined represent the most frequent cause of death in developed nations. The underlying progressive occlusion of large conductance arteries can partially be compensated for by transformation of preexisting collateral arterioles to small artery bypasses, a process referred to as arteriogenesis. Because biomechanical forces have been implicated in the initiation of arteriogenesis, we have investigated the mechanosensitive expression of a pivotal proarteriogenic molecule, monocyte chemoattractant protein (MCP)-1, which governs the recruitment of circulating monocytes to the wall of the remodeling collateral arterioles. Using a new ear artery ligation model and the classic hindlimb ischemia model in mice, we noted that MCP-1 expression is significantly increased in collateral arterioles undergoing arteriogenesis already 24 hours after its onset. By mimicking proarteriogenic perfusion conditions in small mouse arteries, we observed that MCP-1 expression is predominantly upregulated in the smooth muscle cells, which solely sense changes in circumferential wall tension or stretch. Subsequent analyses of cultured endothelial and smooth muscle cells confirmed that cyclic stretch but not shear stress upregulates MCP-1 expression in these cells. Blockade of the mechanosensitive transcription factor activator protein-1 by using a specific decoy oligodeoxynucleotide abolished this stretch-induced MCP-1 expression. Likewise, topical administration of the decoy oligodeoxynucleotide to the mouse ear abrogated arteriogenesis through downregulation of MCP-1 expression and monocyte recruitment. Collectively, these findings point toward a stretch-induced activator protein-1-mediated rise in MCP-1 expression in vascular smooth muscle cells as a critical determinant for the initiation of arteriogenesis.

Adenovirus-mediated kallikrein delivery has been shown to promote blood vessel growth in the limb under both ischemic and normoperfused conditions. Here we investigated whether a continuous supply of kallikrein and kinin peptide can induce neovascularization in a rat model of hindlimb ischemia. Rats underwent femoral artery ligation and localized injection of tissue kallikrein, bradykinin or B1 receptor agonist, followed by infusion of proteins by osmotic minipump. Regional blood flow was monitored weekly by laser Doppler perfusion imaging. Three weeks after surgery, rats receiving kallikrein and kinins showed a significant increase in the perfusion ratio of ischemic vs. normoperfused limb compared to control rats. Similarly, a microsphere assay showed that kallikrein and kinins significantly increased regional blood flow without altering blood pressure. Moreover, kallikrein and kinins significantly augmented capillary and arteriole densities, as quantified by immunostaining with CD-31 and smooth muscle alpha-actin. Both tissue kallikrein and bradykinin increased hemoglobin content in Matrigel implants in mice, providing further evidence of the angiogenic properties. Kinins, when delivered subcutaneously via Matrigel in rats, also increased regional perfusion. This is the first demonstration that local application of tissue kallikrein protein or kinin peptide has therapeutic value in the treatment of ischemic disease by promoting neovascularization.

Previous studies from this laboratory suggest that during juvenile growth, structural changes in the arteriolar network are accompanied by changes in some of the mechanisms responsible for regulation of tissue blood flow. To test the hypothesis that arteriolar myogenic behavior is altered with growth, we studied gracilis muscle arterioles isolated from Sprague-Dawley rats at two ages: 21-28 and 42-49 days. When studied at their respective in vivo pressures, the myogenic index (instantaneous slope of the active pressure-diameter curve) of arterioles from 42-49-day-old rats was more negative than that of arterioles from 21-28-day-old rats, indicating greater myogenic responsiveness. Endothelial denudation, or prostaglandin H(2) (PGH(2))/thromboxane A(2) (TxA(2)) receptor antagonism without denudation, significantly reduced the myogenic responsiveness of arterioles from the older rats over a wide range of pressures but had no consistent effects on the myogenic responsiveness of arterioles from the younger rats. The heme oxygenase inhibitor chromium (III) mesoporphyrin IX chloride had no effect on the myogenic activity of arterioles from either age group. These findings indicate that microvascular growth in young animals is accompanied by an increase in the myogenic behavior of arterioles, possibly because PGH(2) or TxA(2) assumes a role in reinforcing myogenic activity over this period. As a result, the relative contribution of myogenic activity to blood flow regulation in skeletal muscle may increase during rapid juvenile growth.

Objective: The endothelium-dependent dilation of skeletal muscle arterioles is mediated by factors that have not been identified in young rats, and partly mediated by an unidentified hyperpolarizing factor in maturing rats. This study was designed to determine if endogenous hydrogen peroxide (H2O2) contributes to this arteriolar dilation at either of these growth stages., Methods: Gracilis muscle arterioles were isolated from rats at ages 24-26 days ("weanlings") and 46-48 days ("juveniles"). We investigated the effects of catalase treatment on the endothelium-dependent dilation of these vessels to simvastatin and acetylcholine (ACh). Catalase-sensitive 2',7'-dichlorofluorescein (DCF) fluorescence also was measured as an index of H2O2 formation, and arteriolar dilation to exogenous H2O2 was pharmacologically probed in each age group., Results: Responses to simvastatin and ACh were attenuated by catalase in juvenile, but not weanling, arterioles. Juvenile, but not weanling, arterioles also displayed catalase-sensitive DCF fluorescence that was increased by ACh. Exogenous H2O2 could induce dilation in juvenile, but not weanling, arterioles. In juvenile arterioles, this dilation was abolished by the K+ channel inhibitors TEA and glibenclamide, and attenuated by NOS inhibition or endothelial removal., Conclusions: These findings suggest that endogenous H2O2 contributes to endothelium-dependent arteriolar dilation in juvenile rats, but not in younger rats, and that H2O2 acts in juvenile rats by stimulating endothelial NO release and activating smooth muscle K+ channels.

The major vascular endothelial growth factor (VEGF) isoforms are splice variants from a single gene that differ in their extent of heparin affinity due to the absence of the heparin binding domain in the smallest isoform (mouse VEGF120, human VEGF121). A long-held assumption that has guided the use of VEGF isoforms clinically has been that their differences in heparin binding dictate their ability to diffuse through tissue, with VEGF121 moving most freely and that the distribution of recombinant VEGF would have therapeutically relevant consequences. To test this assumption, we delivered the genes encoding these isoforms by myoblast-mediated gene transfer, a means of delivering genes to highly localized sites within muscle. Surprisingly, all isoforms induced comparable extremely localized physiological effects. Significantly, irrespective of the isoform delivered, the vessels passing within several micrometers of muscle fibers expressing VEGF displayed sharply delineated changes in morphology. The induction of capillary wrapping around VEGF-producing fibers, and of vascular malformations in the muscle at high levels, did not differ among isoforms. These results indicate that heparin binding is not essential for the localization of VEGF in adult tissue and suggest that the preferential delivery of VEGF121 cDNA for clinical applications may not have a physiological basis.

Patients with congenital heart disease who have a morphological right ventricle (RV) serving as a systemic ventricle have an increased incidence of RV dysfunction. A different structural response of microvessels to increased pressure load in the RV is a possible mechanism for this dysfunction. To examine the merit of this hypothesis, we explored the possibility that in the normal heart, the branching architecture of microvasculature in walls of the left ventricle (LV) and RV mature differently. The branching structure of intramyocardial arterioles and their downstream branches were investigated using three-dimensional (3D) micro-computed tomography (CT) images in different regions of the RV and LV walls of normal fetal, 1-mo, and 5-mo old pigs. The results point to a significant difference in the volume of myocardium perfused per vessel cross-sectional area (CSA) between the LV and RV walls at 5 mo. We speculate that this difference may be related to the reserve functional capacity of the LV, which requires a corresponding reserve in the expansion capacity of vasculature in the LV wall.

The maladaptive response of the pulmonary vasculature that occurs in patients with congenital diaphragmatic hernia significantly impacts outcome. Muscularized distal pulmonary arterioles inhibit the ability of the neonate to adjust to extrauterine circulation, resulting in severe pulmonary hypertension. This review summarizes the current state of knowledge regarding normal and abnormal development of the lung vascular system and identifies current and potential therapies directed toward preserving or restoring proper pulmonary vascular function.

Stimulation of neovascularization (angiogenesis, arteriogenesis) has emerged as a promising new strategy to treat patients with coronary disease. These strategies aim to improve cardiac function by ensuring myocardial perfusion and to reduce the risk of myocardial infarction. While angiogenesis describes a de-novo formation of small caliber capillary vessels, arteriogenesis leads to the outgrowth of pre-existing arterioles into large conductance collateral arteries. Inflammatory cells (e.g. monocytes), which can produce and secrete growth factors and cytokines, mediate both processes. Several trials have shown that intra-coronary infusion of growth factors or progenitor cells can improve left ventricular function after arterial occlusion. Despite these encouraging results, potential unfavorable effects on plaque progression and stability should not be neglected. Destabilization of atherosclerotic plaques leads to plaque rupture, intravascular thrombosis and tissue infarction. Increased neovascularization of the plaque (e.g. by angiogenesis) is thought to arise from the adventitial vasa vasorum, leading to an abnormal vascular development. This network of immature vessels is a viable source of invading inflammatory cells that can contribute to plaque instability. Furthermore, intra-plaque hemorrhages can lead to accumulation of erythrocyte membranes in the plaque that are rich in phospholipids and free cholesterol, promoting lesion instability through necrotic core expansion. Future angiogenic and arteriogenic approaches need to take these pitfalls into account and should focus on stimulation of vessel growth in combination with neutral or even beneficial effects on plaque formation and composition. This review discusses the delicate balance between the benefits and the drawbacks of therapeutic strategies to influence angiogenesis and arteriogenesis.

Previous studies from this laboratory suggest that during maturation, rapid microvascular growth is accompanied by changes in the mechanisms responsible for regulation of tissue blood flow. To further define these changes, we studied isolated gracilis muscle arterioles from weanling ( approximately 25 days) and juvenile ( approximately 44 days) Sprague-Dawley rats to test the hypothesis that endothelial mechanisms for the control of arteriolar tone are altered with growth. Responses to the endothelium-dependent dilator acetylcholine (ACh) were greater in weanling arterioles (WA) than in juvenile arterioles (JA), whereas there were no consistent differences between age groups in arteriolar responses to other endothelium-dependent agonists (A-23187, vascular endothelial growth factor, and simvastatin). Inhibition of nitric oxide synthase (NOS) with N(omega)-nitro-l-arginine methyl ester (l-NAME) attenuated ACh-induced dilation in JA but not in WA. In JA, combined inhibition of NOS and cyclooxygenase (with indomethacin) reduced the dilator responses to ACh and simvastatin by approximately 90% and approximately 70%, respectively, but had no effect in WA. Cytochrome P450 epoxygenase inhibition [with 2-(propargyloxyphenyl) hexanoic acid] had no effect on responses to ACh or simvastatin in either age group. Inhibition of Ca(2+)-activated or ATP-dependent potassium channels (with tetraethylammonium or glibenclamide, respectively) reduced these arteriolar responses in JA but not those in WA. These findings suggest that in fully grown microvascular networks, endothelium-dependent arteriolar dilation is mediated by the combined release of endothelial nitric oxide and vasodilator prostanoids, and in part through activation of Ca(2+)-activated and ATP-dependent potassium channels. However, during earlier microvascular growth, this dilation is mediated by other factors yet to be identified. This may have significant implications for the regulation of tissue perfusion during microvascular development.

The aim of this work is to investigate the growth of the vasculature in the rat humeral head cartilage after the initial development of the secondary ossification centre until the adult organization. Rats aging from 5 weeks to 12 months were used. Histological observations on humeral heads were implemented with morphometrical analysis. Subsequently, vascular corrosion cast, that permits a three-dimensional observation of the vasculature, were prepared and observed by scanning electron microscopy. In young animals the epiphysis contains thin bone trabeculae and most of the epiphysis is occupied by bone marrow spaces. With age, the bone trabeculae progressively enlarge up to double their thickness. The percentage of bone tissue increases from 33.6 to 58.6% of the entire epiphysis, while the bone marrow spaces tend to increase very little in their mean dimension. Vascular corrosion casts show that the epiphyseal microcirculation is well distinguished from that of the diaphysis, and arises from the vessels present in the capsule and the periosteal networks. In young animals the only capillaries are bone marrow sinusoids and few subchondral capillaries. In adult animals small vessels run between the clusters of sinusoids forming the trabecular circulation. Capillary sprouts from sinusoids are always observed both in the young and adult animals. Thus, in adult rats different proper microcirculatory districts can be distinguished in the epiphysis: (a) the sinusoidal network, that supplies the hematopoiesis of the bone marrow and the adjacent osteogenic tissue; (b) the bone tissue microcirculation, limited to small vessels that supply the metabolism and the remodelling of the bone tissue. The reported microvascular organization and its adaptation to the epiphyseal growth represent the morphological basis for understanding the reciprocal interaction among the different tissues in developing and adult rat epiphysis.

Previous studies have not addressed regional differences in adaptive arteriolar growth in the surviving left ventricular (LV) myocardium after infarction in appropriately aged animals, namely middle-aged or older. Accordingly, we examined the adaptive postinfarction growth of arterioles in two distinct regions, i.e., the LV free wall (LVFW) and septum, of middle-aged rats. We induced a myocardial infarction (MI) in 12-mo-old rats to analyze 1) protein expression in VEGF/Flt-1/Flk-1 and angiopoietin (Ang)-1/Ang-2/Tie-2 systems, 2) the arteriolar DNA synthesis, 3) the extent of the arteriolar bed, and 4) the alteration in minimal coronary vascular resistance. In both regions, arteriolar DNA synthesis was activated between days 4 and 7 after MI. Whereas in the LVFW the degree of DNA synthesis declined between days 11 and 14 post-MI, it continued to rise in the septum, and at day 14, the percentage of the arterioles undergoing DNA synthesis was comparable in the LVFW and the septum (9.7 +/- 1.6 and 7 +/- 2.1%, respectively). Arteriolar DNA synthesis was mainly associated with upregulation of Ang-2 and Tie-2 in both LV regions. Although 4 wk after MI the arteriolar beds in the LVFW and the septum expanded to the size of sham-operated rats, this growth did not compensate for the greater minimal coronary vascular resistance in the former. Thus our findings suggest that 1) the dynamics in adaptive arteriolar growth were similar between the two regions, despite a delay in the septum; and 2) the perfusion deficit in post-MI rats cannot be accounted for by inadequate adaptive growth of arterioles.

Background and Purpose: Reactive oxygen species are believed to be an important determinant of vascular growth. We examined effects of genetic deficiency of copper-zinc superoxide dismutase (CuZnSOD; SOD1) on structure and function of cerebral arterioles., Methods: Systemic arterial pressure (SAP) and cross-sectional area of the vessel wall (CSA) and superoxide (O2-) levels (relative fluorescence of ethidium [ETH]) were examined in maximally dilated cerebral arterioles in mice with targeted disruption of one (+/-) or both (-/-) genes encoding CuZnSOD. Wild-type littermates served as controls. Vasodilator responses were tested in separate groups of mice., Results: CSA and ETH were significantly increased (P<0.05) in both CuZnSOD+/- and CuZnSOD-/- mice (CSA=435+/-24 and 541+/-48 microm2; ETH=18+/-1 and 34+/-2%) compared with wild-type mice (CSA=327+/-28 microm2; ETH=6%). Furthermore, the increases in CSA and ETH relative to wild-type mice were significantly greater (P<0.05) in CuZnSOD-/- mice than in CuZnSOD+/- mice (CSA=108 versus 214 microm2; ETH=12 versus 28%). In addition, dilatation of cerebral arterioles in response to acetylcholine, but not nitroprusside, was reduced by approximately 25% in CuZnSOD+/- (P<0.075) and 50% in CuZnSOD-/- mice (P<0.05) compared with wild-type mice., Conclusions: Cerebral arterioles in CuZnSOD+/- and CuZnSOD-/- mice undergo marked hypertrophy. These findings provide the first direct evidence in any blood vessel that CuZnSOD normally inhibits vascular hypertrophy suggesting that CuZnSOD plays a major role in regulation of cerebral vascular growth. The findings also suggest a gene dosing effect of CuZnSOD for increases in O2-, induction of cerebral vascular hypertrophy and impaired endothelium-dependent dilatation.

Objective: Exercise training has been shown to increase regional blood flow capacity to muscle tissue containing fibers that experience increased activity during exercise. The purpose of this study was to test the hypothesis that the increased blood flow capacity is partially the result of increases in arteriolar density (number of arterioles/mm2 of tissue), specifically in skeletal muscle tissue, with the largest relative increase in muscle fiber activity during training bouts., Methods: This hypothesis was tested by comparing and contrasting the effects of endurance exercise training (ET) and interval sprint training (IST) on arteriolar density in soleus muscle (S) red (Gr) and white (Gw) portions of gastrocnemius muscle of male Sprague Dawley rats. ET rats completed 10 weeks of treadmill training 30 m/min, 15% grade, 60 min/day, 5 days/week, while IST rats completed 10 weeks of IST consisting of six 2.5-min exercise bouts, with 4.5-min rest between bouts (60 m/min, 15% incline), 5 days/week. The hypothesis would be supported if ET increased arteriolar density in S and Gr and if IST increased arteriolar density in Gw., Results: ET increased arteriolar density above values of sedentary rats (SED) in both the Gw (ET = 0.93 +/- 0.19 arterioles/microm2; SED = 0.44 +/- 0.09 arterioles/microm2) and Gr (ET = 0.97 +/- 0.1 arterioles/microm2; SED = 0.51 +/- 0.06 arterioles/microm2) muscles, but not in S (ET = 1.69 +/- 0.45 arterioles/microm2; SED = 1.51 +/- 0.34 arterioles/microm2) muscle. In contrast, IST did not alter arteriolar density in Gw or Gr muscle tissue. Although arterial wall thickness was greater in S (3.95 +/- 0.40 microm) and Gr (6.24 +/- 0.59 microm) than Gw (2.76 +/- 0.18 microm), neither ET or IST altered mean wall thickness in either muscle., Conclusion: Increases in blood flow capacity produced in Gr and Gw by ET appear to be due in part to increased arteriolar density. In contrast, increased arteriolar density does not contribute to increased blood flow capacity of Gw in IST rats.

Objectives: The purpose of this study was to investigate whether a novel fibroblast growth factor-2 gene formulation, providing a localized and sustained availability of the adenoviral vector from a collagen-based matrix, in combination with CO 2 transmyocardial laser revascularization would lead to an enhanced angiogenic response and improved myocardial function., Methods: Fibroblast growth factor-2 gene was delivered by means of an adenoviral vector (adenoviral fibroblast growth factor-2) formulated in a collagen-based matrix. The ischemic areas of 33 animals were then treated. Group 1 was treated with CO 2 transmyocardial laser revascularization; group 2 was treated with intramyocardial injections of adenoviral fibroblast growth factor-2 in a collagen-based matrix; group 3 had a combination treatment of matrix adenoviral fibroblast growth factor-2 and CO 2 transmyocardial laser revascularization; and group 4 received injections with saline-formulated adenoviral fibroblast growth factor-2. Baseline left ventricular function was assessed by echocardiography and cine magnetic resonance imaging. Studies were repeated 6 weeks after treatment. Vascular development was assessed using anti-alpha-actin immunohistochemistry., Results: Matrix adenoviral fibroblast growth factor-2 + transmyocardial laser revascularization-treated areas had a 105% increase in arteriolar development versus either treatment alone ( P < .05) and a 390% increase compared with saline-formulated adenoviral fibroblast growth factor-2 treatment alone ( P < .05). Contractility was significantly improved in matrix adenoviral fibroblast growth factor-2 + transmyocardial laser revascularization-treated areas as measured by myocardial wall thickening. This functional improvement was confirmed by cine magnetic resonance imaging, in which a 90% increase in the contractility of the treated segments was demonstrated after matrix adenoviral fibroblast growth factor-2 + transmyocardial laser revascularation. The other treatments provided significantly less restoration of myocardial function., Conclusions: The increase in angiogenesis as a result of matrix adenoviral fibroblast growth factor-2 gene therapy in combination with CO 2 transmyocardial laser revascularization is greater than that seen in either therapy alone. A concomitant improvement in myocardial function was seen as a result of this angiogenic response.

Background: Acidic fibroblast growth factor (FGF-1) has been identified as a potent mitogen for vascular cells, inducing formation of mature blood vessels in vitro and in vivo and represents one of the most promising approaches for the treatment of ischemic cardiovascular diseases by gene therapy. Nevertheless, and most probably due to the few experimental models able to address the issue, no study has described the therapeutic effects of FGF-1 gene transfer in subjects with peripheral arterial disease (PAD) exhibiting a clinically relevant cardiovascular pathology., Methods: In order to assess the potency of FGF-1 gene transfer for therapeutic angiogenesis in ischemic skeletal muscles displaying decreased gene expression levels and sustained impaired formation of collateral vessels and arterioles, we developed a model of PAD in hamsters with a background of hypercholesterolemia. Hamsters fed a cholesterol-rich diet and subjected to hindlimb ischemia exhibit a sustained impaired angiogenic response, as evidenced by decreased angiographic score and histological quantification of arterioles in the ischemic muscles., Results: In this model, we demonstrate that NV1FGF (a human FGF-1 expression plasmid), given intramuscularly 14 days after induction of hindlimb ischemia, promoted the formation of both collateral vessels and arterioles 14 days after treatment (i.e. 28 days post-ischemia)., Conclusions: Our data provide evidence that NV1FGF can reverse the cholesterol-induced impairment of revascularization in a hamster model of hindlimb ischemia by promoting the growth of both collateral vessels and arterioles in ischemic muscles exhibiting significantly decreased levels of gene expression compared with control muscles. Therefore, this study underscores the relevance of NV1FGF gene therapy to overcome perfusion defects in patients with PAD., (Copyright 2004 John Wiley & Sons, Ltd.)

Myocardial infarction (MI) is characterized by ventricular remodeling, hypertrophy of the surviving myocardium, and an insufficient angiogenic response. Thyroxine is a powerful stimulus for myocardial angiogenesis. Male rats that underwent coronary artery ligation and subsequent MI were given 3,5-diiodothyropropionic acid (DITPA; MI+DITPA group) during a 3-wk period. We evaluated ventricular remodeling using echocardiography and histology and myocardial vessel growth using image analysis. Protein expression was assessed using Western blotting and immunohistochemistry. This study tested the hypothesis that the thyroxine analog DITPA facilitates angiogenesis and influences postinfarction remodeling in the surviving hypertrophic myocardium. The increase in the region of akinesis (infarct expansion) was blunted in the MI+DITPA rats compared with the MI group (3 vs. 21%); the treated rats had smaller percent increases in the left ventricular (LV) volume (64 +/- 14 vs. 95 +/- 12) and the LV volume-to-mass ratio (47 +/- 13 vs. 84 +/- 10) as well as a blunted decrease in ejection fraction (-9 +/- 8 vs. -30 +/- 7%). Arteriolar length density was higher after treatment in the largest (>50% of the free wall) infarcts (64 +/- 3 vs. 43 +/- 7). Angiogenic growth factors [vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)] and the angiopoietin receptor tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (Tie-2) values were elevated during the first week after infarction. DITPA did not cause additional increases in VEGF or Tie-2 values but did induce an increase in bFGF value after 3 days of treatment. This study provides the first evidence for an anatomical basis, i.e., attenuated ventricular remodeling and arteriolar growth, for improved function attributed to DITPA therapy of the infarcted heart. The favorable influences of DITPA on LV remodeling after large infarction are principally due to border zone preservation.

Diabetes macro- and microvascular disease causes tissue hypoperfusion. This deficit, together with a failure to mount an adequate angiogenic response, might explain why vascular occlusion evolves more severely among diabetic patients. The present study investigated whether prophylactic gene therapy with human tissue kallikrein (hTK) may protect diabetic limbs from the consequences of supervening ischemia. Vehicle (saline) or an adenovirus carrying the gene for either hTK (Ad.hTK) or luciferase (Ad.Luc) was injected into left adductor muscles of streptozotocin-induced type 1 diabetic mice 2 weeks before operative occlusion of the ipsilateral femoral artery. Saline-injected nondiabetic mice served as controls. Hindlimb blood flow recovery was analyzed sequentially over the 2 weeks after ischemia induction. At necroscopy, microvessel density and endothelial cell proliferation and apoptosis were quantified in skeletal muscles. We found that limb perfusion recovery of saline-injected type 1 diabetic mice is delayed because of insufficient reparative neovascularization and excessive activation of endothelial cell apoptosis. By contrast, prophylactic Ad.hTK renewed the ability to mount an appropriate neovascularization response to ischemia, suppressed apoptosis, and upregulated endothelial nitric oxide synthase expression. Ultimately, correction of diabetic endotheliopathy by Ad.hTK allowed proper perfusion recovery as seen in nondiabetic mice. These discoveries disclose new therapeutic options for the treatment of diabetic complications.

Growth of collateral vessels is potentially able to preserve structure and a variable degree of function in subtended tissues in the presence of arterial occlusions. The process of transformation of a small arteriole into much larger conductance artery is called arteriogenesis. Small arterioles that interconnect side branches proximal from the arterial occlusion with distal ones experience increased fluid shear stress because of the increased blood flow velocity attributable to the pressure gradient along the bridging collaterals. This activates the endothelium and leads to monocyte adhesion and infiltration with the subsequent production of growth factors and proteases. Preexistent arterioles are essential. Their presence is genetically determined. Arteriogenesis is not organ- or species-specific; coronary or peripheral collateral vessels develop following the same design principles in mice, rats, rabbits, or dogs. In contrast to angiogenesis, arteriogenesis is not dependent on the presence of hypoxia/ischemia.

We have shown previously that implantation of myoblasts constitutively expressing the VEGF-A gene into nonischemic mouse skeletal muscle leads to overgrowth of capillary-like blood vessels and hemangioma formation. These aberrant effects occurred directly at the implantation site. We show here that these regions result from angiogenic capillary growth and involve a change in capillary growth pattern and that smooth muscle-coated vessels similar to arterioles form directly adjacent to the implantation site. Myoblasts genetically engineered to produce VEGF were implanted into mouse leg muscles. Implantation sites were surrounded by a zone of dense capillary-sized vessels, around which was a second zone of muscle containing larger, smooth-muscle-covered vessels but few capillaries, and an outer zone of muscle exhibiting normal capillary density. The lack of capillaries in the middle region suggests that the preexisting capillaries adjacent to the implantation site underwent enlargement and/or fusion and recruited a smooth muscle coat. Capillaries at the implantation site were frequently wrapped around VEGF-producing muscle fibers and were continuous with the circulation and were not observed to include bone-marrow-derived endothelial cells. In contrast with the distant arteriogenesis resulting from VEGF delivery described in previous studies, we report here that highly localized arterioles also form adjacent to the site of delivery.

Aim: To investigate the changes of microvascular architecture, ultrastructure and permeability of rat jejunal villi at different ages., Methods: Microvascular corrosion casting, scanning electron microscopy, transmission electron microscopy and Evans blue infiltration technique were used in this study., Results: The intestinal villous plexus of adult rats consisted of arterioles, capillary network and venules. The marginal capillary extended to the base part of the villi and connected to the capillary networks of adjacent villi. In newborn rats, the villous plexus was rather simple, and capillary network was not formed. The villous plexus became cone-shaped and was closely arrayed in ablactation rats. In adult rats, the villous plexus became tongue-shaped and was enlarged both in height and width. In aged rats, the villous plexus shrank in volume and became shorter and narrower. The diametral ratio of villous arteriole to villous venule increased as animals became older. The number of endothelial holes, the thickness of basal membrane and the permeability of microvasculature were increased over the entire course of development from newborn period to aged period., Conclusion: The digestive and absorptive functions of the rat jejunum at different ages are highly dependent upon the state of villous microvascular architecture and permeability, and blood circulation is enhanced by collateral branches such as marginal capillary, through which blood is drained to the capillary networks of adjacent villi.

The blood supply to the retina and the lens in 32 gerbils (Meriones unguiculatus) of both sexes from infancy to maturity was studied under light and stereoscopic microscopes, and a scanning electron microscope. Mercox (CL-2R; Dai Nippon Ink, Tokyo, Japan) was injected into the left ventricle of 30 animals in order to visualize the blood supply to the retina and the lens from the ophthalmic artery. The central retinal artery arises from the ophthalmic artery, passes through the papilla of the optic nerve together with the central retinal vein and penetrates the vitreous space (cavity of the eye) between the lens and the internal limiting membrane of the retina, where it divides into the central branches covering the lens and the parietal branches to supply the retina. The former passes through the hyaloid space after branching several arterioles and then covers the lens like a network from its medial and marginal sides. Different from small experimental animals, the parietal branches, just after separating from the central one, divides into the nasal, dorsal and temporal branches in the vitreous space, each of which then subdivides to distribute across the retina on the inner limiting membrane, then delineates the membrana vasculosa retinae. This basal pattern of vasculization 1 day after birth continues to death. Both the central and parietal branches of the central retinal artery correspond to the branches of the hyaloid artery in embryo and the latter is preserved in adult gerbils.

The role of the collateral circulation has been controversially discussed over many decades. With the availability of purified growth factors such as the fibroblast growth factors several studies provided data, that the growth of collateral arteries, termed arteriogenesis, is not limited to its natural timecourse. When applied in experimental models FGF in particular led to a significant increase in collateral conductance upon arterial occlusion. Thus the proliferation of preexisting bypassing arterioles could be enhanced therapeutically. The purpose of this review is to discuss the physiological importance of different kinds of vascular growth (e.g. vasculogenesis, angiogenesis, arteriogenesis) to enhance blood flow to ischemic limbs or the heart. It is outlined that large conductance arteries rather than capillary networks are needed to compensate for perfusion deficits due to atherosclerotic stenosis or occlusion.

Previous studies from our laboratory and those of others have shown thyroxine to be a stimulator of coronary microvascular growth. The present study tested the hypothesis that 3,5-diiodothyropropionic acid (DITPA), a thyroid hormone analog with inotropic but not chronotopic characteristics, is angiogenic in the nonischemic heart. Daily injections (3.75 mg/kg sc) of DITPA to Sprague-Dawley rats affected protein increases in vascular endothelial growth factor (VEGF)(164), VEGF(188,) basic fibroblast growth factor (bFGF) (FGF-2), angiopoietin-1, and Tie-2 during the first few days of treatment. After 3 wk of treatment, arteriolar length density and the relative number of terminal arterioles (<10 microm diameter) increased in the left ventricle as determined by image analysis of perfuse-fixed hearts. These findings occurred in hearts that did not undergo changes in mass nor in increases in capillary length density. We conclude that DITPA, which is known to improve ventricular function after infarction, is angiogenic in normal nonischemic hearts.

Background: The neurotrophin nerve growth factor (NGF) regulates neuron survival and differentiation. Implication in neovascularization is supported by statement of NGF and its high-affinity receptor at vascular level and by NGF property of stimulating vascular endothelial cell proliferation. The present study investigated the involvement of endogenous NGF in spontaneous reparative response to ischemia. Mechanisms and therapeutic potential of NGF-induced neovascularization were examined., Methods and Results: Unilateral limb ischemia was produced in CD1 mice by femoral artery resection. By ELISA and immunohistochemistry, we documented that statement of NGF and its high-affinity receptor is upregulated in ischemic muscles. The functional relevance of this phenomenon was assessed by means of NGF-neutralizing antibody. Chronic NGF blockade abrogated the spontaneous capillarization response to ischemia and augmented myocyte apoptosis. Then we tested whether NGF administration may exert curative effects. Repeated NGF injection into ischemic adductors increased capillary and arteriole density, reduced endothelial cell and myofiber apoptosis, and accelerated perfusion recovery, without altering systemic hemodynamics. In normoperfused muscles, NFG-induced capillarization was blocked by vascular endothelial growth factor-neutralizing antibodies, dominant-negative Akt, or NO synthase inhibition., Conclusions: These results indicate that NGF plays a functional role in reparative neovascularization. Furthermore, supplementation of the growth factor promotes angiogenesis through a vascular endothelial growth factor-Akt-NO-mediated mechanism. In the setting of ischemia, potentiation of NGF pathway stimulates angiogenesis and arteriogenesis, thereby accelerating hemodynamic recovery. NGF might be envisaged as a utilitarian target for the treatment of ischemic vascular disease.

Background: The application of ultrasound to microbubbles in skeletal muscle creates capillary ruptures. We tested the hypothesis that this bioeffect could be used to stimulate the growth and remodeling of new arterioles via natural repair processes, resulting in an increase in skeletal muscle nutrient blood flow., Methods and Results: Pulsed ultrasound (1 MHz) was applied to exposed rat gracilis muscle after intravenous microbubble injection. Capillary rupturing was visually verified by the presence of red blood cells in the muscle, and animals were allowed to recover. Ultrasound-microbubble-treated and contralateral sham-treated muscles were harvested 3, 7, 14, and 28 days later. Arterioles were assessed by smooth muscle alpha-actin staining, and skeletal muscle blood flow was measured with 15- micro m fluorescent microspheres. An approximately 65% increase in arterioles per muscle fiber was noted in treated muscles compared with paired sham-treated control muscles at 7 and 14 days after treatment. This increase in arterioles occurred across all studied diameter ranges at both 7 and 14 days after treatment. Arterioles per muscle fiber in sham-treated and untreated control muscles were comparable, indicating that the surgical intervention itself had no significant effect. Hyperemia nutrient blood flow in treated muscles was increased 57% over that in paired sham-treated control muscles., Conclusions: Capillary rupturing via microbubble destruction with ultrasound enhances arterioles per muscle fiber, arteriole diameters, and maximum nutrient blood flow in skeletal muscle. This method has the potential to become a clinical tool for stimulating blood flow to organs affected by occlusive vascular disease.

Objective: To investigate the potential influence of hemodynamic stresses on the development of the arcade arteriole (AA) network during normal maturation., Methods: AA network data were collected from ink-filled Wistar-Kyoto rat gracilis muscles and used to construct hemodynamic computational models of the AA network at 7 (WKY(7)) and 13 (WKY(13)) weeks of age., Results: Mean coefficients of variation for pressure, circumferential wall stress, and wall shear stress were 0.13, 0.12, and 0.48, respectively. Wall shear rate variability across bifurcations generated deviations in mean energy cost that were 9-30% above theoretical minimum, with many bifurcations exhibiting substantially higher energy costs. With the exception of the lowest pressure AA segments, the monotonic relationship between wall shear stress and pressure in the AAs was nearly identical from 7 to 13 weeks of age., Conclusions: Low coefficients of variation for computed AA pressures indicate that an even pressure head is maintained over the muscle during remodeling of the AA network. The anastomotic structure of the network creates high shear rate variability that, in turn, creates high-energy costs in some regions of the network. The results are consistent with the hypothesis that, during development, the maintenance of mean circumferential wall stress and the pressure-shear stress relationship are operative design principles for collateral arteriole development.