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Background: Endothelial cells (ECs) use glycolysis to produce energy. In preclinical models of peripheral arterial disease, further activation of EC glycolysis was ineffective or deleterious in promoting hypoxia-dependent angiogenesis, whereas pentose phosphate pathway activation was effective. Hexosamine biosynthesis pathway, pentose phosphate pathway, and glycolysis are closely linked. Glucosamine directly activates hexosamine biosynthesis pathway., Methods: Hind-limb ischemia in endothelial nitric oxide synthase knockout (eNOS -/- ) and BALB/c mice was used. Glucosamine (600 μg/g per day) was injected intraperitoneally. Blood flow recovery was assessed using laser Doppler perfusion imaging and angiogenesis was studied by CD31 immunostaining. In vitro, human umbilical vein ECs and mouse microvascular ECs with glucosamine, L-glucose, or vascular endothelial growth factor (VEGF 165 a) were tested under hypoxia and serum starvation. Cell Counting Kit-8, tube formation, intracellular reactive oxygen species, electric cell-substrate impedance sensing, and fluorescein isothiocyanate dextran permeability were assessed. Glycolysis and oxidative phosphorylation were assessed by seahorse assay. Gene expression was assessed using RNA sequencing, real-time quantitative polymerase chain reaction, and Western blot. Human muscle biopsies from patients with peripheral arterial disease were assessed for EC O-GlcNAcylation before and after supervised exercise versus standard medical care., Results: On day 3 after hind-limb ischemia, glucosamine-treated versus control eNOS -/- mice had less necrosis (n=4 or 5 per group). Beginning on day 7 after hind-limb ischemia, glucosamine-treated versus control BALB/c mice had higher blood flow, which persisted to day 21, when ischemic muscles showed greater CD31 staining per muscle fiber (n=8 per group). In vitro, glucosamine versus L-glucose ECs showed improved survival (n=6 per group) and tube formation (n=6 per group). RNA sequencing of glucosamine versus L-glucose ECs showed increased amino acid metabolism (n=3 per group). That resulted in increased oxidative phosphorylation (n=8-12 per group) and serine biosynthesis pathway without an increase in glycolysis or pentose phosphate pathway genes (n=6 per group). This was associated with better barrier function (n=6-8 per group) and less reactive oxygen species (n=7 or 8 per group) compared with activating glycolysis by VEGF 165 a. These effects were mediated by activating transcription factor 4, a driver of exercise-induced angiogenesis. In muscle biopsies from humans with peripheral arterial disease, EC/O-GlcNAcylation was increased by 12 weeks of supervised exercise versus standard medical care (n=6 per group)., Conclusions: In cells, mice, and humans, activation of hexosamine biosynthesis pathway by glucosamine in peripheral arterial disease induces an "exercise-like" angiogenesis and offers a promising novel therapeutic pathway to treat this challenging disorder., Competing Interests: None.

Background In endothelial cells (ECs), glycolysis, regulated by PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase, isoform-3), is the major metabolic pathway for ATP generation. In preclinical peripheral artery disease models, VEGF 165 a (vascular endothelial growth factor 165 a) and microRNA-93 both promote angiogenesis. Methods and Results Mice following hind-limb ischemia (HLI) and ECs with, and without, hypoxia and serum starvation were examined with, and without, microRNA-93 and VEGF 165 a. Post-HLI perfusion recovery was monitored. EC metabolism was studied using seahorse assay, and the expression and activity of major metabolism genes were assessed. Reactive oxygen species levels and EC permeability were evaluated. C57Bl/6J mice generated a robust angiogenic response to HLI, with ECs from ischemic versus nonischemic muscle demonstrating no increase in glycolysis. Balb/CJ mice generated a poor angiogenic response post-HLI; ischemic versus nonischemic ECs demonstrated significant increase in glycolysis. MicroRNA-93-treated Balb/CJ mice post-HLI showed better perfusion recovery, with ischemic versus nonischemic ECs showing no increase in glycolysis. VEGF 165 a-treated Balb/CJ mice post-HLI showed no improvement in perfusion recovery with ischemic versus nonischemic ECs showing significant increase in glycolysis. ECs under hypoxia and serum starvation upregulated PFKFB3. In ECs under hypoxia and serum starvation, VEGF 165 a versus control significantly upregulated PFKFB3 and glycolysis, whereas miR-93 versus control demonstrated no increase in PFKFB3 or glycolysis. MicroRNA-93 versus VEGF 165 a upregulated glucose-6-phosphate dehydrogenase expression and activity, activating the pentose phosphate pathway. MicroRNA-93 versus control increased reduced nicotinamide adenine dinucleotide phosphate and virtually eliminated the increase in reactive oxygen species. In ECs under hypoxia and serum starvation, VEGF 165 a significantly increased and miR-93 decreased EC permeability. Conclusions In peripheral artery disease, activation of the pentose phosphate pathway to promote angiogenesis may offer potential therapeutic advantages.

The vascular endothelial injury occurs in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, but the mechanisms are poorly understood. We sought to determine the frequency and type of cytokine elevations and their relationship to endothelial injury induced by plasma from patients with SARS-CoV-2 versus controls. Plasma from eight consecutively enrolled patients hospitalized with acute SARS-CoV-2 infection was compared to controls. Endothelial cell (EC) barrier integrity was evaluated using ECIS (electric cell-substrate impedance sensing) on human lung microvascular EC. Plasma from all SARS-CoV-2 but none from controls decreased transendothelial resistance to a greater degree than that produced by tumor necrosis factor-alpha (TNF-α), the positive control for the assay. Thrombin, angiopoietin 2 (Ang2), and vascular endothelial growth factor (VEGF), complement factor C3a and C5a, and spike protein increased endothelial permeability, but to a lesser extent and a shorter duration when compared to SARS-CoV-2 plasma. Analysis of Ang2, VEGF, and 15 cytokines measured in plasma revealed striking patient-to-patient variability within the SARS-CoV-2 patients. Pretreatment with thrombin inhibitors, single, or combinations of neutralizing antibodies against cytokines, Ca3 and C5a receptor antagonists, or with ACE2 antibody failed to lessen the SARS-CoV-2 plasma-induced EC permeability. The EC barrier destructive effects of plasma from patients with SARS-CoV-2 were susceptible to heat inactivation. Plasma from patients hospitalized with acute SARS-CoV-2 infection uniformly disrupts lung microvascular integrity. No predicted single, or set of, cytokine(s) accounted for the enhanced vascular permeability, although the factor(s) were heat-labile. A still unidentified but potent circulating factor(s) appears to cause the EC disruption in SARS-CoV-2 infected patients. IMPORTANCE Lung vascular endothelial injury in SARS-CoV-2 patients is one of the most important causes of morbidity and mortality and has been linked to more severe complications including acute respiratory distress syndrome (ARDS) and subsequent death due to multiorgan failure. We have demonstrated that in eight consecutive patients with SARS-CoV-2, who were not selected for evidence of endothelial injury, the diluted plasma-induced intense lung microvascular damage, in vitro . Known endothelial barrier-disruptive agents and proposed mediators of increased endothelial permeability in SARS-CoV-2, induced changes in permeability that were smaller in magnitude and shorter in duration than plasma from patients with SARS-CoV-2. The effect on endothelial cell permeability of plasma from patients with SARS-CoV-2 was heat-labile. The main plasma factor that causes the increased endothelial permeability remains to be identified. Our study provides a possible approach for future studies to understand the underlying mechanisms leading to vascular injury in SARS-CoV-2 infections.

The objective was to evaluate the effect of photobiomodulation (PBM) using 980 nm diode laser therapy (0.60 W, 0.77 W cm -2 , 36 J, 46 J cm -2 , 60 s) irradiated in continuous wave mode by flat-top hand-piece on socket healing in the maxilla and mandible. A split-mouth experimental design was performed on 6 dogs. The 3 rd premolar tooth was extracted from the maxilla and mandibles for both sides. The right-sided sockets were irradiated (PBM group), and the left-sided sockets were kept as control. Irradiation was done after extraction and at 48-h interval for 14 days. Both the buccal and lingual sides were irradiated to reach a total irradiation time of 120 s. Bone density was evaluated at 3, 4 and 5 weeks using cone beam computed tomography. We showed that maxillary sockets in the PBM group had higher bone density compared to control one at 3, 4, 5 weeks (P = 0.029, <0.001, <0.001), respectively. Mandibular sockets revealed no significant difference between PBM and control at 3 weeks (P = 0.347), while at 4 and 5 weeks PBM group showed higher bone density (P = 0.004, <0.001). In both groups, there was a significant increase (P < 0.001) in bone density by time which was higher in the PBM group. We concluded that PBM using a flat-top hand-piece of 980-nm improved the bone density of extraction sockets., (© 2020 American Society for Photobiology.)

Purpose: The aim of this study was to test the hypothesis that the dose of estrogen replacement therapy may have an influence on the clinical, radiographic, and histopathologic changes within the temporomandibular joint (TMJ)., Materials and Methods: A prospective experimental study was conducted in 12 mature ovariectomized dogs. Dogs were randomly allocated into 1 of 4 groups (OVX-E0, dogs that did not receive any estrogen replacement therapy; OVX-E0.5, dogs that received 0.15 mg/kg of estradiol; OVX-E1, dogs that received 0.3 mg/kg of estradiol; and OVX-E2, dogs that received 0.6 mg/kg of estradiol); dogs were evaluated clinically for 12 weeks; and contact radiographic and histopathologic examinations of the TMJ were performed just after euthanasia., Results: Radiographic examination of the TMJ in the OVX-E0 group showed narrowing of the joint space with marginal osteophyte formation along the mandibular condyle. The OVX-E0.5 group showed mild widening of the joint space with no remarkable changes within the mandibular fossa or condyle. The OVX-E1 group was free of radiographic changes within the TMJ. High doses of estrogen in the OVX-E2 group showed marked flattening of the mandibular condyle and fossa with sclerosis of the subchondral bone. Histopathologic sections in the OVX-E0 group showed thin compact bone with scanty, less organized lacunae. The OVX-E0.5 group showed compact bone of medium thickness with large osteons and disorganized lacunae. The OVX-E1 group showed thick compact bone with reduced intertubercular spaces and organized lacunae. The OVX-E2 group showed thin bone with reduced trabecular and increased intertrabecular thickness. The collagen content did not change significantly among the 4 groups, whereas its quality changed significantly (P < .05)., Conclusions: Estrogen dosage is potentially a key regulator of bone metabolism within the TMJ. Estrogen replacement therapy exhibited an inverted U-shaped beneficial effect. Estrogen depletion as well as high doses of estrogen resulted in clinical, radiographic, and histopathologic changes within the TMJ. Estrogen replacement therapy should be prescribed at the optimum dose when indicated as hormonal replacement therapy., (Copyright © 2020 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

Nivolumab, a monoclonal antibody against programmed cell death-1 receptor, is increasingly used in advanced cancers. While nivolumab use enhances cancer therapy, it is associated with increased immune-related adverse events. We describe an elderly man who presented in ketoacidosis after receiving nivolumab for metastatic renal cell carcinoma. On presentation, he was hyperpneic and laboratory analyses showed hyperglycemia and anion-gapped metabolic acidosis consistent with diabetic ketoacidosis. No other precipitating factors, besides nivolumab, were identified. Pre-nivolumab blood glucose levels were normal. The patient responded to treatment with intravenous fluids, insulin and electrolyte replacement. He was diagnosed with insulin-dependent autoimmune diabetes mellitus secondary to nivolumab. Although nivolumab was stopped, he continued to require multiple insulin injection therapy till his last follow-up 7 months after presentation. Clinicians need to be alerted to the development of diabetes mellitus and diabetic ketoacidosis in patients receiving nivolumab., Learning Points: Diabetic ketoacidosis should be considered in the differential of patients presenting with metabolic acidosis following treatment with antibodies to programmed cell death-1 receptor (anti-PD-1).Autoimmune islet cell damage is the presumed mechanism for how insulin requiring diabetes mellitus can develop de novo following administration of anti-PD-1.Because anti-PD-1 works by the activation of T-cells and reduction of 'self-tolerance', other autoimmune disorders are likely to be increasingly recognized with increased use of these agents.

Twenty suckled CharloixxHereford beef cows (5 cows/group) were assigned at random to receive 100 microg GnRH (IM) at either 2 to 3, 7 to 8, 15 to 16, or 31 to 32 days postpartum, Groups 1 through 4, respectively. Blood samples for hormone determinations were collected at time 0 (pre-GnRH), every half hr for 3 hr, and at 4.0 hr and 6.0 hr post-GnRH. Mean plasma LH, estradiol-17beta, or progesterone concentrations were not different among groups prior to GnRH. Plasma LH increased (P<.05) following GnRH in Groups 2, 3 and 4, but not in Group 1. Peak GnRH induced LH release was greater (P<.05) in Groups 3 and 4 than in Groups 1 or 2. Correlation coefficients between days postpartum and peak LH release (r=.72), and estradiol-17beta concentrations and time of LH peak (r=-.42) were significant (P<.05). These data indicate that LH release in response to GnRH, in suckled beef cows is not fully restored until 15 to 16 days postpartum.

The objectives of the experiment were to evaluate the efficacy of using progesterone concentrations in milk and palpation per rectum on days 21 or 22 postbreeding to estimate pregnancy and evaluate management practices; and to investigate physiological occurrences leading to incorrect diagnosis of pregnancy when serial samples of milk were collected. Of particular interest were indications of early embyronic death and insemination of cows not in estrus. Milk samples were collected at the afternoon milking of days 0 or 1 (day 0 = day of estrus), 9 or 10, 21 or 22 and 27 or 28 following breeding in 200 lactating dairy cows. Tentative diagnosis of pregnancy was made based on concentrations of progesterone in milk on days 21 and 22 alone and on days 21 or 22 and 27 or 28. In addition all cows were palpated per rectum on days 21 or 22 postbreeding and a tentative pregnancy diagnosis was made. Pregnancy was confirmed by examination of the genital tract per rectum between 35 and 50 days after breeding. Values of 4 ng/ml or greater and/or the presence of a mature corpus luteum were considered positive signs of pregnancy. Progesterone in milk ranged from 0.1 to 18 ng/ml. On days 0 or 1, 9 or 10, 21 or 22 and 27 or 28 concentrations of progesterone in milk averaged 1.5 +/- 0.3, 11.1 +/- 0.5, 12.0 +/- 0.4 12.5 +/- 0.5 ng/ml for pregnant cows. Corresponding samples from nonpregnant cows averaged 1.2 +/- 0.2, 10.3 +/- 0.4, 3.0 +/- 0.4, 6.8 +/- 0.6 ng/ml, respectively. Ninety-six and 104 cows were classified as pregnant and nonpregnant on days 21 or 22 as compared to 78 and 118 cows diagnosed as pregnant and nonpregnant on days 21 or 22 and 27 or 28 combined. Pregnancy detection by progesterone in milk on days 21 or 22 with pregnancy determined via rectal palpation 35 to 50 days postbreeding was 77 and 100% accurate for positive and negative diagnosis, respectively. The percent agreement using progesterone in milk on days 21 or 22 and 27 or 28 combined was 95 and 100%, respectively, for positive and negative diagnosis. Diagnosis based on rectal palpation 21 or 22 days postbreeding was 63 92 (69%) and 76 88 (87%) for pregnant and nonpregnant cows, respectively. Ten of the 200 cows had progesterone concentratins in milk of > 4 ng/ml at the time of breeding. Six of these cows were pregnant from a previous insemination. The other four cows were nonpregnant and were inseminated during the luteal phase of the cycle. In conclusion, measurement of progesterone in milk is a useful tool in early detection of pregnant and nonpregnant cows and may be useful in detecting reproductive problems in a dairy herd. It will probably be most useful when used in combination with later pregnancy diagnosis per rectum .

Twenty-seven dairy cows were evenly assigned to one of three groups and given an intramuscular injection of 2 mg estradiol benzoate. Cows in group 1 were greater than 30 days postpartum at treatment and had been diagnosed via rectal palpation to have ovarian cysts. Cows in groups 2 and 3 were 12 to 14 and 30 to 40 days postpartum, respectively. Blood plasma was collected from all cows before treatment and then every three hours for 36 hours post-treatment. Concentrations of LH, estradiol-17 beta and progesterone in plasma were determined by radioimmunoassay. Four, zero and five cows in groups 1, 2 and 3, respectively, had concentrations of progesterone greater than 1.0 ng/ml before estradiol benzoate treatment. None of these cows had a peak LH release greater than 5 ng/ml following estradiol benzoate treatment. The numbers of cows with progesterone concentrations less than 1 ng/ml that released LH (>5 ng/ml) in response to estradiol benzoate were 3 of 5, 3 of 9, and 4 of 4 for groups 1, 2, and 3, respectively; the proportion for group 3 was higher (P<.05) than for group 2. Of the cows that released LH, mean peak LH concentrations were 33.3+/-5.4, 14.8+/-7.2 and 24.6+/-9.8 ng/ml for groups 1, 2 and 3, respectively, and the duration of the LH increase was 8.0+/-1.0, 8.0+/-2.0 and 13.0+/-4.0 hours. The time from estradiol benzoate treatment to peak LH release for cows with ovarian cysts (25+/-2 hours) was delayed (P<.05) compared with that for cows 30 to 40 days postpartum without ovarian cysts (16+/-1 hour). In summary, responsiveness to estradiol benzoate is regained between 2 to 4 weeks postpartum in most cows. In addition, some cows with ovarian cysts can release LH in response to estradiol benzoate, but peak LH release is delayed compared to cows at a comparable stage postpartum without ovarian cysts.

The objective was to determine the relationship of prior follicular development to GnRH-induced cyclic ovarian activity in dairy cows postpartum. Sixty lactating Holstein and Guernsey cows from the University of Missouri dairy herds were assigned at random to one of two groups. Group I consisted of 20 cows (control) given a single intramuscular (IM) injection of saline. Group II consisted of 40 cows given a single injection of 100 micrograms gonadotropin releasing hormone (GnRH) IM. Treatments were administered 12 to 14 days postpartum. All cows were palpated per rectum to monitor ovarian activity prior to treatment (time 0) and 2 to 3 and 7 to 9 days post-treatment. Blood was collected via tail vessel puncture at time 0 and 7 to 9 days post-treatment. The percentage of cows exhibiting a follicle 10 mm or greater in diameter prior to treatment was not different between Group I (50%) and Group II (43%). However, twenty (50%) of the GnRH-treated cows initiated cyclic ovarian activity following treatment as evidenced by palpable CL and plasma progesterone greater than or equal to 1.0 ng/ml on day 9 post-treatment in contrast to only four (20%) saline-treated cows (P less than 0.05). Seventeen of 20 cows ovulating in Group II exhibited a palpable follicle greater than or equal to 10 mm in diameter prior to treatment compared to 3 of 4 cows in Group I. Prior to treatment, plasma concentrations of LH, estradiol-17 beta and progesterone were not different (P less than .05) between cows in Groups I and II. In Group II, preinjection levels of estradiol-17 beta and LH for cows ovulating in response to GnRH were higher (P less than .01) than in cows not ovulating. Postinjection concentrations of progesterone in plasma on days 7 to 9 were higher (P less than .01) in cows ovulating than in cows not ovulating. Six (30%) cows in Group I developed ovarian cysts prior to conception as compared to 12.5% (5) of the cows in Group II (P less than .05). The present study suggests: (1) GnRH treatment as early as 12 to 14 days postpartum may initiate cyclic ovarian activity in normal dairy cows; (2) elevated preinjection concentrations of estradiol-17 beta and follicular growth are important for GnRH induced ovulations; and (3) GnRH treatment 12 to 14 days postpartum may be useful in reducing abnormal ovarian activity.