Leptin is a physiological regulator of skeletal muscle angiogenesis and is locally produced by PDGFRα and PDGFRß expressing perivascular cells.

Skeletal muscle capillarity is characteristically reduced in mature leptin receptor-deficient (Leprdb) mice, which has been attributed to the capillary loss that occurs secondary to metabolic dysfunction. Despite wide recognition of leptin as a pro-angiogenic molecule, the contribution of this adipokine has largely been overlooked in peripheral tissues. Moreover, prior documentation of leptin production within skeletal muscle indicates a potential paracrine role in maintaining local tissue homeostasis. Thus, we hypothesized that leptin is a physiological local paracrine regulator of skeletal muscle angiogenesis and that its production may be modulated by nutrient availability. Leprdb mice exhibited impaired angiogenesis during normal developmental maturation of skeletal myocytes, corresponding with an inability to increase vascular endothelial growth factor-A (VEGFA) mRNA and protein levels between 4 and 13 weeks. In cultured murine and human skeletal myocytes, recombinant leptin increased VEGFA mRNA levels. Leptin mRNA was detectable in skeletal muscle, increasing with prolonged high-fat feeding in mice, and with adiposity in human subjects. Platelet-derived growth factor receptor (PDGFR)α- and PDGFRß- expressing perivascular cell populations were identified as leptin producing within skeletal muscle of mice and humans. Furthermore, in response to 2 weeks of high-fat feeding, PDGFRß+ but not PDGFRα+ cells increased leptin production. We conclude that leptin is a physiological regulator of the capillary network in skeletal muscle and stimulates VEGFA production by skeletal myocytes. PDGFRß expressing perivascular cells exhibit the capacity to act as local "nutrient-sensors" that couple nutrient status to leptin production in skeletal muscle.

Tissue Inhibitor of Metalloproteinase 1 Influences Vascular Adaptations to Chronic Alterations in Blood Flow.

Remodeling of the skeletal muscle microvasculature involves the coordinated actions of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs). We hypothesized that the loss of TIMP1 would enhance both ischemia and flow-induced vascular remodeling by increasing MMP activity. TIMP1 deficient (Timp1-/- ) and wild-type (WT) C57BL/6 mice underwent unilateral femoral artery (FA) ligation or were treated with prazosin, an alpha-1 adrenergic receptor antagonist, in order to investigate vascular remodeling to altered flow. Under basal conditions, Timp1-/- mice had reduced microvascular content as compared to WT mice. Furthermore, vascular remodeling was impaired in Timp1-/- mice. Timp1-/- mice displayed reduced blood flow recovery in response to FA ligation and no arteriogenic response to prazosin treatment. Timp1-/- mice failed to undergo angiogenesis in response to ischemia or prazosin, despite maintaining the capacity to increase VEGF-A and eNOS mRNA. Vascular permeability was increased in muscles of Timp1-/- mice in response to both prazosin treatment and FA ligation, but this was not accompanied by greater MMP activity. This study highlights a previously undescribed integral role for TIMP1 in both vascular network maturation and adaptations to ischemia or alterations in flow. J. Cell. Physiol. 232: 831-841, 2017. © 2016 Wiley Periodicals, Inc.

Metabolic effects of prazosin on skeletal muscle insulin resistance in glucocorticoid-treated male rats.

High-dose glucocorticoids (GC) induce skeletal muscle atrophy, insulin resistance, and reduced muscle capillarization. Identification of treatments to prevent or reverse capillary rarefaction and metabolic deterioration caused by prolonged elevations in GCs would be therapeutically beneficial. Chronic administration of prazosin, an α1-adrenergic antagonist, increases skeletal muscle capillarization in healthy rodents and, recently, in a rodent model of elevated GCs and hyperglycemia. The purpose of this study was to determine whether prazosin administration would improve glucose tolerance and insulin sensitivity, through prazosin-mediated sparing of capillary rarefaction, in this rodent model of increased GC exposure. Prazosin was provided in drinking water (50 mg/l) to GC-treated or control rats (400 mg implants of either corticosterone or a wax pellet) for 7 or 14 days (n = 5-14/group). Whole body measures of glucose metabolism were correlated with skeletal muscle capillarization (C:F) at 7 and 14 days in the four groups of rats. Individual C:F was found to be predictive of insulin sensitivity (r2 = 0.4781), but not of glucose tolerance (r2 = 0.1601) and compared with water only, prazosin treatment decreased insulin values during oral glucose challenge by approximately one-third in corticosterone (Cort)-treated animals. Cort treatment, regardless of duration, induced significant glycolytic skeletal muscle atrophy (P < 0.05), decreased IRS-1 protein content (P < 0.05), and caused elevations in FOXO1 protein expression (P < 0.05), which were unaffected with prazosin administration. In summary, it appears that α1-adrenergic antagonism improves Cort-induced skeletal muscle vascular impairments and reduces insulin secretion during an oral glucose tolerance test, but is unable to improve the negative alterations directly affecting the myocyte, including muscle size and muscle signaling protein expression.

Endothelial FoxO proteins impair insulin sensitivity and restrain muscle angiogenesis in response to a high-fat diet.

Skeletal muscle microvascular dysfunction contributes to disease severity in type 2 diabetes. Recent studies indicate a role for Forkhead box O (FoxO) transcription factors in modulating endothelial cell phenotype. We hypothesized that a high-fat (HF) diet generates a dysfunctional vascular niche through an increased expression of endothelial FoxO. FoxO1 protein increased (+130%) in the skeletal muscle capillaries from HF compared to normal chow-fed mice. FoxO1 protein was significantly elevated in cultured endothelial cells exposed to the saturated fatty acid palmitate or the proinflammatory cytokine TNF-α. In HF-fed mice, endothelium-directed depletion of FoxO1/3/4 (FoxO(Δ)) improved insulin sensitivity (+110%) compared to that of the controls (FoxO(L/L)). The number of skeletal muscle capillaries increased significantly in the HF-FoxO(Δ) mice. Transcript profiling of skeletal muscle identified significant increases in genes associated with angiogenesis and lipid metabolism in HF-FoxO(Δ) vs. HF-FoxO(L/L) mice. HF-FoxO(Δ) muscle also was characterized by a decrease in inflammation-related genes and an enriched M2 macrophage signature. We conclude that endothelial FoxO proteins promote insulin resistance in HF diet, which may in part result from FoxO proteins establishing an antiangiogenic and proinflammatory microenvironment within skeletal muscle. These findings provide mechanistic insight into the development of microvascular dysfunction in the progression of type 2 diabetes.-Nwadozi, E., Roudier, E., Rullman, E., Tharmalingam, S., Liu, H.-Y., Gustafsson, T., Haas, T. L. Endothelial FoxO proteins impair insulin sensitivity and restrain muscle angiogenesis in response to a high-fat diet.

Forkhead BoxO transcription factors restrain exercise-induced angiogenesis.

The physiological process of exercise-induced angiogenesis involves the orchestrated upregulation of angiogenic factors together with repression of angiostatic factors. The Forkhead Box 'O' (FoxO) transcription factors promote an angiostatic environment in pathological contexts. We hypothesized that endothelial FoxO1 and FoxO3a also play an integral role in restricting the angiogenic response to aerobic exercise training. A single exercise bout significantly increased levels of FoxO1 and FoxO3a mRNA (5.5- and 1.7-fold, respectively) and protein (1.7- and 2.2-fold, respectively) within the muscles of mice 2 h post-exercise compared to sedentary. Training abolished the exercise-induced increases in both FoxO1 and FoxO3a mRNA and proteins, and resulted in significantly lower nuclear levels of FoxO1 and FoxO3a protein (0.5- and 0.4-fold, respectively, relative to sedentary). Thrombospondin 1 (THBS1) protein level closely mirrored the expression pattern of FoxO proteins. The 1.7-fold increase in THBS1 protein following acute exercise no longer occurred after 10 days of repeated exercise. Endothelial cell-directed conditional deletion of FoxO1/3a/4 in mice prevented the increase in THBS1 mRNA following a single exercise bout. Mice harbouring the endothelial FoxO deletion also demonstrated a significant 20% increase in capillary to muscle fibre ratio after only 7 days of training while 14 days of training was required to elicit a similar increase in wildtype littermates. Our results demonstrate that the downregulation of FoxO1 and FoxO3a proteins facilitates angiogenesis in response to repeated exercise. In conclusion, FoxO proteins can delay exercise-induced angiogenesis, and thus are critical regulators of the physiological angiogenic response in skeletal muscle.

Endothelial cell TIMP-1 is upregulated by shear stress via Sp-1 and the TGFß1 signaling pathways.

Laminar shear stress promotes vascular integrity by inhibiting proteolysis of the extracellular matrix (ECM) surrounding the microvasculature. We hypothesized that the matrix metalloproteinase inhibitor TIMP-1 would be upregulated in endothelial cells exposed to shear stress. Microvascular endothelial cells isolated from rat or mouse skeletal muscles were exposed to laminar shear stress for 2, 4, or 24 h. A biphasic increase in TIMP-1 protein was observed at 2 and 24 h of shear stress exposure. Sp-1 siRNA prevented the increase in TIMP-1 after 2, but not 24, hours of shear exposure. TGFß production and Smad2/3 phosphorylation are increased by shear stress. Inhibition of TGFß signaling, either by use of the TGFß receptor 1 inhibitor SB-431542 or with Smad 2/3 siRNA, abrogated the shear stress-induced increase in TIMP-1 mRNA after 24 h of shear stress exposure. These results suggest that both acute and chronic elevated laminar shear stress act to maintain vessel integrity through increasing TIMP-1 production, but that the TGFß signaling pathway is essential to maintain TIMP-1 expression during chronic shear stress.

Shaping and remodeling of the fetoplacental circulation: aspects of health and disease.

Extensive vascular adaptations occur during pregnancy, and these result in the formation of a low-resistance placental circulation that maintains high blood flow to the developing fetus. These adaptations encompass both functional and structural alterations, including altered vasoreactivity of resistance vessels, arterial remodeling and angiogenesis. This Special Topics issue presents a collection of expert reviews that summarize the current state of knowledge on the regulation of the structural and functional changes that occur within the fetoplacental circulation, as well as introduce emerging research questions and tools. Emphasis is placed on defining the mechanisms that underlie these physiological adaptations, as a foundation for applying this knowledge to the development of improved early detection markers and treatments for pathological conditions such as preeclampsia, gestational diabetes mellitus, and fetal growth restriction.

Endothelial FoxO1 is an intrinsic regulator of thrombospondin 1 expression that restrains angiogenesis in ischemic muscle.

Peripheral artery disease (PAD) is characterized by chronic muscle ischemia. Compensatory angiogenesis is minimal within ischemic muscle despite an increase in angiogenic factors. This may occur due to the prevalence of angiostatic factors. Regulatory mechanisms that could evoke an angiostatic environment during ischemia are largely unknown. Forkhead box O (FoxO) transcription factors, known to repress endothelial cell proliferation in vitro, are potential candidates. Our goal was to determine whether FoxO proteins promote an angiostatic phenotype within ischemic muscle. FoxO1 and the angiostatic matrix protein thrombospondin 1 (THBS1) were elevated in ischemic muscle from PAD patients, or from mice post-femoral artery ligation. Mice with conditional endothelial cell-directed deletion of FoxO proteins (Mx1Cre (+), FoxO1,3,4 (L/L) , referred to as FoxOΔ) were used to assess the role of endothelial FoxO proteins within ischemic tissue. FoxO deletion abrogated the elevation of FoxO1 and THBS1 proteins, enhanced hindlimb blood flow recovery and improved neovascularization in murine ischemic muscle. Endothelial cell outgrowth from 3D explant cultures was more robust in muscles derived from FoxOΔ mice. FoxO1 overexpression induced THBS1 production, and a direct interaction of endogenous FoxO1 with the THBS1 promoter was detectable in primary endothelial cells. We provide evidence that FoxO1 directly regulates THBS1 within ischemic muscle. Altogether, these findings bring novel insight into the regulatory mechanisms underlying the repression of angiogenesis within peripheral ischemic tissues.

Transcriptomic profiling reveals sex-specific molecular signatures of adipose endothelial cells under obesogenic conditions.

Female mice display greater adipose angiogenesis and maintain healthier adipose tissue than do males upon high-fat diet feeding. Through transcriptome analysis of endothelial cells (EC) from the white adipose tissue of male and female mice high-fat-fed for 7 weeks, we found that adipose EC exhibited pronouncedly sex-distinct transcriptomes. Genes upregulated in female adipose EC were associated with proliferation, oxidative phosphorylation, and chromatin remodeling contrasting the dominant enrichment for genes related to inflammation and a senescence-associated secretory of male EC. Similar sex-biased phenotypes of adipose EC were detectable in a dataset of aged EC. The highly proliferative phenotype of female EC was observed also in culture conditions. In turn, male EC displayed greater inflammatory potential than female EC in culture, based on basal and tumor necrosis factor alpha-stimulated patterns of gene expression. Our study provides insights into molecular programs that distinguish male and female EC responses to pathophysiological conditions.

Identification of a mechanism underlying regulation of the anti-angiogenic forkhead transcription factor FoxO1 in cultured endothelial cells and ischemic muscle.

Chronic limb ischemia, a complication commonly observed in conjunction with cardiovascular disease, is characterized by insufficient neovascularization despite the up-regulation of pro-angiogenic mediators. One hypothesis is that ischemia induces inhibitory signals that circumvent the normal capillary growth response. FoxO transcription factors exert anti-proliferative and pro-apoptotic effects on many cell types. We studied the regulation of FoxO1 protein in ischemic rat skeletal muscle following iliac artery ligation and in cultured endothelial cells. We found that FoxO1 expression was increased in capillaries within ischemic muscles compared with those from rats that underwent a sham operation. This finding correlated with increased expression of p27(Kip1) and reduced expression of Cyclin D1. Phosphorylated Akt was reduced concurrently with the increase in FoxO1 protein. In skeletal muscle endothelial cells, nutrient stress as well as lack of shear stress stabilized FoxO1 protein, whereas shear stress induced FoxO1 degradation. Endogenous FoxO1 co-precipitated with the E3 ubiquitin ligase murine double minute-2 (Mdm2) in endothelial cells, and this interaction varied in direct relation to the extent of Akt and Mdm2 phosphorylation. Moreover, ischemic muscles had a decreased level of Mdm2 phosphorylation and a reduced interaction between Mdm2 and FoxO1. Our results provide novel evidence that the Akt-Mdm2 pathway acts to regulate endothelial cell FoxO1 expression and illustrate a potential mechanism underlying the pathophysiological up-regulation of FoxO1 under ischemic conditions.

Angiogenesis within the duodenum of patients with cirrhosis is modulated by mechanosensitive Kruppel-like factor 2 and microRNA-126.

BACKGROUND: The mechanism involved in neovascularization in splanchnic circulation and the main trigger that induces angiogenesis in patients with cirrhosis are not fully recognized. AIMS: To explore the involvement of flow sensitive lung Kruppel-like factor (KLF2), microRNA-126 (miR-126), angiopoietin-2 (Ang-2) and heme oxygenase-1 (HO-1) in modulation of vascular endothelial growth factor (VEGF) signalling that have a critical effect on growth of new blood vessels. METHODS: Duodenal biopsies from 22 patients with cirrhosis and 10 controls were obtained during routine endoscopy. The process of angiogenesis was evaluated by a measurement of CD31 concentration, immunodetection of CD34 protein and estimation of capillary densities. Messenger RNA (mRNA) and protein expressions were analysed by real-time PCR, Western blot or ELISA respectively. RESULTS: Markers of angiogenesis (both, CD31 and CD34) were significantly enhanced in cirrhotic patients. In comparison to healthy controls, levels of Ang-2 and KLF-2 mRNAs as well as Ang-2, KLF-2, HO-1, VEGF protein expressions were considerably increased. Levels of sCD163, a surrogate marker of portal hypertension, correlated with levels of Ang-2, (P = 0.021) and VEGF (P = 0.009). The expression of miR-126, a KLF2-mediated regulator of the VEGF signalling was enhanced in cirrhotic patients. CONCLUSIONS: Our results demonstrate, for the first time in humans, that neovascularization is induced in duodenal tissue of patients with cirrhosis and proangiogenic factors such as KLF-2, Ang-2, miR-126 and VEGF can contribute to the angiogenesis induced by hemodynamic forces. Thus, cirrhosis-induced blood flow and pressure within splanchnic vessels may be important hemodynamic triggers that initiate the angiogenic signalling cascade.

Quantitative Methods to Assess Adipose Vasculature.

Adipose tissue depots are invested with an extensive capillary network that is closely associated with maintenance of adipose functions and enables healthy tissue expansion. The capillary network displays a high level of plasticity, demonstrating either growth (angiogenesis) or regression (rarefaction) under various physiological/pathological conditions, which has significant consequences for cardiometabolic health. Thus, the visualization and quantification of adipose vascular networks is an important aspect of studying factors that regulate adipose tissue health. This chapter provides an overview of several methods to quantify adipose vascularization. In-depth protocols are provided for the visualization of vascular structures by staining and imaging of whole-mount adipose tissues or paraffin-embedded adipose tissue sections, together with the quantitative analysis of vascularization from these images.

Scientific meeting report: International Biochemistry of Exercise 2022.

Exercise is one of the only nonpharmacological remedies known to counteract genetic and chronic diseases by enhancing health and improving life span. Although the many benefits of regular physical activity have been recognized for some time, the intricate and complex signaling systems triggered at the onset of exercise have only recently begun to be uncovered. Exercising muscles initiate a coordinated, multisystemic, metabolic rewiring, which is communicated to distant organs by various molecular mediators. The field of exercise research has been expanding beyond the musculoskeletal system, with interest from industry to provide realistic models and exercise mimetics that evoke a whole body rejuvenation response. The 18th International Biochemistry of Exercise conference took place in Toronto, Canada, from May 25 to May 28, 2022, with more than 400 attendees. Here, we provide an overview of the most cutting-edge exercise-related research presented by 66 speakers, focusing on new developments in topics ranging from molecular and cellular mechanisms of exercise adaptations to exercise therapy and management of disease and aging. We also describe how the manipulation of these signaling pathways can uncover therapeutic avenues for improving human health and quality of life.

Exploring risk factors at the molecular level.

Risk factors for cardiovascular diseases trigger molecular changes that harm the endothelial cells in the heart, but exercise can suppress these effects.

High Glucose Treatment Limits Drosha Protein Expression and Alters AngiomiR Maturation in Microvascular Primary Endothelial Cells via an Mdm2-dependent Mechanism.

Diabetes promotes an angiostatic phenotype in the microvascular endothelium of skeletal muscle and skin. Angiogenesis-related microRNAs (angiomiRs) regulate angiogenesis through the translational repression of pro- and anti-angiogenic genes. The maturation of micro-RNA (miRs), including angiomiRs, requires the action of DROSHA and DICER proteins. While hyperglycemia modifies the expression of angiomiRs, it is unknown whether high glucose conditions alter the maturation process of angiomiRs in dermal and skeletal muscle microvascular endothelial cells (MECs). Compared to 5 mM of glucose, high glucose condition (30 mM, 6-24 h) decreased DROSHA protein expression, without changing DROSHA mRNA, DICER mRNA, or DICER protein in primary dermal MECs. Despite DROSHA decreasing, high glucose enhanced the maturation and expression of one angiomiR, miR-15a, and downregulated an miR-15a target: Vascular Endothelial Growth Factor-A (VEGF-A). The high glucose condition increased Murine Double Minute-2 (MDM2) expression and MDM2-binding to DROSHA. Inhibition of MDM2 prevented the effects evoked by high glucose on DROSHA protein and miR-15a maturation in dermal MECs. In db/db mice, blood glucose was negatively correlated with the expression of skeletal muscle DROSHA protein, and high glucose decreased DROSHA protein in skeletal muscle MECs. Altogether, our results suggest that high glucose reduces DROSHA protein and enhances the maturation of the angiostatic miR-15a through a mechanism that requires MDM2 activity.

p38 MAPK activity is stimulated by vascular endothelial growth factor receptor 2 activation and is essential for shear stress-induced angiogenesis.

Increased capillary shear stress induces angiogenesis in skeletal muscle, but the signaling mechanisms underlying this response are not known. We hypothesize that shear stress-dependent activation of vascular endothelial growth factor receptor 2 (VEGFR2) causes p38 and ERK1/2 phosphorylation, which contribute to shear stress-induced angiogenesis. Skeletal muscle microvascular endothelial cells were sheared (12 dynes/cm(2), 0.5-24 h). VEGFR2-Y1214 phosphorylation increased in response to elevated shear stress and VEGF stimulation. p38 and ERK1/2 phosphorylation increased at 2 h of shear stress but only p38 remained phosphorylated at 6 and 24 h of shear stress. VEGFR2 inhibition abrogated p38, but not ERK1/2 phosphorylation. VEGF production was increased in response to shear stress at 6 h, and this increased production was abolished by p38 inhibition. Male Sprague-Dawley rats were administered prazosin (50 mg/L drinking water, 1, 2, 4, or 7 days) to induce chronically elevated capillary shear stress in skeletal muscle. In some experiments, mini-osmotic pumps were used to dispense p38 inhibitor SB203580 or its inactive analog SB202474, to the extensor digitorum longus (EDL) of control and prazosin-treated rats. Immunostaining and Western blotting showed increases in p38 phosphorylation in capillaries from rats treated with prazosin for 2 days but returned to basal levels at 4 and 7 days. p38 inhibition abolished the increase in capillary to muscle fiber ratio seen after 7 days of prazosin treatment. Our data suggest that p38 activation is necessary for shear stress-dependent angiogenesis.

The angiogenic response to skeletal muscle overload is not dependent on mast cell activation.

OBJECTIVE: To determine if mast cell activation in skeletal muscle contributes to overload-induced angiogenesis. METHODS: Extensor digitorum longus muscle was overloaded through extirpation of the synergist muscle tibialis anterior. Muscles were removed after 1, 2, 4, 7 or 14 days, and mast cell density and degranulation were quantified by histology. The mast cell stabilizer, cromolyn, was administered acutely or chronically to test if mast cell degranulation contributes to overload-induced angiogenesis. Angiogenesis was determined by calculating capillary to muscle Fiber ratio; mast cell density and activation were quantified by histology, MMP-2 levels were assessed by gelatin zymography and VEGF protein levels were assessed by Western blotting. RESULTS: Muscle overload increased mast cell degranulation and total mast cell number within 7 days. Mast cell stabilization with cromolyn attenuated degranulation but did not inhibit the increased mast cell density, MMP-2 activity, VEGF protein levels or the increase in capillary number following muscle overload. CONCLUSIONS: Mast cell degranulation and accumulation precede overload-induced angiogenesis, but mast cell activation is not critical to the angiogenic response following skeletal muscle overload.

Metabolic Coordination of Pericyte Phenotypes: Therapeutic Implications.

Pericytes are mural vascular cells found predominantly on the abluminal wall of capillaries, where they contribute to the maintenance of capillary structural integrity and vascular permeability. Generally quiescent cells in the adult, pericyte activation and proliferation occur during both physiological and pathological vascular and tissue remodeling. A considerable body of research indicates that pericytes possess attributes of a multipotent adult stem cell, as they are capable of self-renewal as well as commitment and differentiation into multiple lineages. However, pericytes also display phenotypic heterogeneity and recent studies indicate that lineage potential differs between pericyte subpopulations. While numerous microenvironmental cues and cell signaling pathways are known to regulate pericyte functions, the roles that metabolic pathways play in pericyte quiescence, self-renewal or differentiation have been given limited consideration to date. This review will summarize existing data regarding pericyte metabolism and will discuss the coupling of signal pathways to shifts in metabolic pathway preferences that ultimately regulate pericyte quiescence, self-renewal and trans-differentiation. The association between dysregulated metabolic processes and development of pericyte pathologies will be highlighted. Despite ongoing debate regarding pericyte classification and their functional capacity for trans-differentiation in vivo, pericytes are increasingly exploited as a cell therapy tool to promote tissue healing and regeneration. Ultimately, the efficacy of therapeutic approaches hinges on the capacity to effectively control/optimize the fate of the implanted pericytes. Thus, we will identify knowledge gaps that need to be addressed to more effectively harness the opportunity for therapeutic manipulation of pericytes to control pathological outcomes in tissue remodeling.

High-fat diet pre-conditioning improves microvascular remodelling during regeneration of ischaemic mouse skeletal muscle.

AIM: Critical limb ischaemia (CLI) is characterized by inadequate angiogenesis, arteriolar remodelling and chronic myopathy, which are most severe in type 2 diabetic patients. Hypertriglyceridaemia, commonly observed in these patients, compromises macrovascular function. However, the effects of high-fat diet-induced increases in circulating lipids on microvascular remodelling are not established. Here, we investigated if high-fat diet would mimic the detrimental effect of type 2 diabetes on post-ischaemia vascular remodelling and muscle regeneration, using a mouse model of hindlimb ischaemia. METHODS: Male C57Bl6/J mice were fed with normal or high-fat diets for 8 weeks prior to unilateral femoral artery ligation. Laser doppler imaging was used to assess limb perfusion recovery. Vascular recovery, inflammation, myofibre regeneration and fibrosis were assessed at 4 or 14 days post-ligation by histology and RNA analyses. Capillary-level haemodynamics were assessed by intravital microscopy of control and regenerating muscles 14 days post-ligation. RESULTS: High-fat diet increased muscle succinate dehydrogenase activity and capillary-level oxygen supply. At 4 days post-ligation, no diet differences were detected in muscle damage, inflammatory infiltration or capillary activation. At 14 days post-ligation, high fat-fed mice displayed accelerated limb blood flow recovery, elevated capillary and arteriole densities as well as greater red blood cell supply rates and capillary-level oxygen supply. Regenerating muscles from high fat-fed mice displayed lower interstitial fat and collagen deposition. CONCLUSION: The muscle-level adaptations to high-fat diet improved multiple aspects of muscle recovery in response to ischaemia and did not recapitulate the worse outcomes seen in diabetic CLI patients.

Differential role of beta-catenin in VEGF and histamine-induced MMP-2 production in microvascular endothelial cells.

Increases in endothelial cell permeability and production of matrix-degrading enzymes are two early steps in the angiogenic process. Factors such as vascular endothelial growth factor (VEGF) and histamine induce the angiogenic process through alterations in both permeability and proteolysis. We hypothesized that beta-catenin acts as a positive regulator of MMP-2 and MT1-MMP transcription following VEGF or histamine stimulation. Rat microvascular endothelial cells were exposed to VEGF or histamine overnight and MMP-2 protein production was assessed by gelatin zymography. Latent MMP-2 protein levels were increased following VEGF and histamine treatment as were MMP-2 mRNA transcript levels. Endothelial cells exposed to VEGF and histamine had an increased level of nuclear beta-catenin, which was sensitive to inhibition of the PI3-kinase signaling pathway. Promoter assays indicated increased transcriptional activity of both MMP-2 and MT1-MMP in endothelial cells co-transfected with luciferase reporter constructs and beta-catenin. Inhibition of beta-catenin signaling with inhibitor of catenin and T cell factor (ICAT) revealed that the VEGF-induced increase in MMP-2 mRNA is beta-catenin dependent. Interestingly, while MMP-2 mRNA levels increased in response to histamine H1 or H2 receptor activation, significantly larger increases were observed in cells co-treated with ICAT and histamine or the histamine receptor agonists, HTMT and dimaprit. While both VEGF and histamine increase nuclear beta-catenin and MMP-2 production, the role of beta-catenin in MMP-2 regulation differs between the two stimuli.