Search

Exercise is one of the only non-pharmacologic 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 signalling system 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, including extracellular vesicles such as exosomes. 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. This year's 18th International Biochemistry of Exercise conference took place in Toronto, Canada, from May 25th to May 28th, 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, diabetes, and aging. We also provide descriptions on how manipulation of these signaling pathways provide therapeutic avenues for improving human health and quality of life.

Capillaries are composed of endothelial cells that are partially wrapped by a mural cell known as a pericyte. There is growing interest in the functional roles of pericytes within the skeletal muscle microcirculation and how they might ultimately influence skeletal muscle functions. Generally, pericytes are thought to stabilize the capillary structure, modify endothelial cell functions, and participate in the process of new capillary formation, angiogenesis. Based on their structural organization and phenotypic behaviors, pericytes are postulated to perform a broad set of roles within the skeletal muscle that may include coordinating tissue homeostasis in healthy muscle and during the regeneration processes in damaged muscle. These functions indicate potential therapeutic opportunities targeting or utilizing pericytes to improve muscle health under pathophysiological conditions such as diabetes or muscle ischemia. This review will discuss the current knowledge of skeletal muscle pericyte phenotype and function and the evidence supporting the influence of pericytes in skeletal muscle health under physiological and pathological states.

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.

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.

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.

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 PDGFRβ- 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, PDGFRβ+ 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. PDGFRβ expressing perivascular cells exhibit the capacity to act as local "nutrient-sensors" that couple nutrient status to leptin production in skeletal muscle.

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.

Impaired angiogenesis is a hallmark of metabolically dysfunctional adipose tissue in obesity. However, the underlying mechanisms restricting angiogenesis within this context remain ill-defined. Here, we demonstrate that induced endothelial-specific depletion of the transcription factor Forkhead Box O1 (FoxO1) in male mice led to increased vascular density in adipose tissue. Upon high-fat diet feeding, endothelial cell FoxO1-deficient mice exhibited even greater vascular remodeling in the visceral adipose depot, which was paralleled with a healthier adipose tissue expansion, higher glucose tolerance and lower fasting glycemia concomitant with enhanced lactate levels. Mechanistically, FoxO1 depletion increased endothelial proliferative and glycolytic capacities by upregulating the expression of glycolytic markers, which may account for the improvements at the tissue level ultimately impacting whole-body glucose metabolism. Altogether, these findings reveal the pivotal role of FoxO1 in controlling endothelial metabolic and angiogenic adaptations in response to high-fat diet and a contribution of the endothelium to whole-body energy homeostasis., eLife digest In the body, thread-like blood vessels called capillaries weave their way through our tissues to deliver oxygen and nutrients to every cell. When a tissue becomes bigger, existing vessels remodel to create new capillaries that can reach far away cells. However, in obesity, this process does not happen the way it should: when fat tissues expand, new blood vessels do not always grow to match. The starved fat cells can start to dysfunction, which causes a range of issues, from inflammation and scarring of the tissues to problems with how the body processes sugar and even diabetes. Yet, it is still unclear why exactly new capillaries fail to form in obesity. What we know is that a protein called FoxO (short for Forkhead box O) is present in the cells that line the inside of blood vessels, and that it can stop the development of new capillaries. FoxO controls how cells spend their energy, and it can force them to go into a resting state. During obesity, the levels of FoxO actually increase in capillary cells. Therefore, it may be possible that FoxO prevents new blood vessels from growing in the fat tissues of obese individuals. To find out, Rudnicki et al. created mice that lack the FoxO protein in the cells lining the capillaries, and then fed the animals a high-fat diet. These mutant mice had more blood vessels in their fat tissue, and their fat cells looked healthier. They also stored less fat than normal mice on the same diet, and their blood sugar levels were normal. This was because the FoxO-deprived cells inside capillaries were burning more energy, which they may have obtained by pulling sugar from the blood. These results show that targeting the cells that line capillaries helps new blood vessels to grow, and that this could mitigate the health problems that arise with obesity, such as high levels of sugar (diabetes) and fat in the blood. However, more work is needed to confirm that the same cellular processes can be targeted to obtain positive health outcomes in humans.

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.

Background: Impaired capillary growth (angiogenesis) in skeletal muscle and adipose tissue contributes to the development of metabolic disorders in obese males. This association remains unexplored in females, despite mounting evidence that endothelial cells have sex-specific transcriptional profiles. Therefore, herein we assessed whether males and females show distinct angiogenic capacities in response to diet-induced obesity. Methods: Age-matched male and female mice were fed normal chow or high-fat obesogenic diets for 16 weeks. At the end of diet period, systemic glucose disposal was assessed as well as insulin sensitivity of skeletal muscle and visceral adipose tissue. Capillary content and the expression of angiogenic regulators were also evaluated in these tissues. Results: When placed on a high-fat diet, female mice gained less weight than males and showed a metabolic phenotype similar to NC-fed mice, contrasting with the impaired whole-body glucose metabolism observed in high-fat-fed males. However, high-fat-feeding elevated serum lipid levels similarly in male and female mice. Although skeletal muscle of high-fat-fed female mice had higher insulin sensitivity than male counterparts, no sex difference was detected in muscle capillarization. Metabolic functions of perigonadal white adipose tissue (pgWAT) were retained in high-fat-fed females, as evidenced by smaller adipocytes with preserved insulin sensitivity, greater responsiveness to isoproterenol, higher expression of Adiponectin and a lower ratio of Leptin:Adiponectin mRNA. An enhanced browning phenotype was detected in HF-fed female adipocytes with upregulation of Ucp1 expression. PgWAT from high-fat-fed females also showed augmented capillary number and expression of endothelial cell markers, which was associated with elevated mRNA levels of pro-angiogenic mediators, including vascular endothelial growth factor A (Vegfa) and its receptor (Vegfr2), the Notch ligand Jagged-1 (Jag1) and Angiopoietin-2 (Angpt2). Conclusion: Taken together, our findings provide novel evidence that visceral adipose tissue of female mice display greater levels of pro-angiogenic factors and vascularity than males in response to high-fat diet. This phenotype is associated with preserved metabolic homeostasis at both tissue and systemic levels. Our study discloses that a thus-far-unappreciated sex-specific difference in the regulation of adipose angiogenesis may contribute to an individual's susceptibility to developing adipose dysfunction and obesity-related metabolic disturbances.

We demonstrated in a previous study that murine double minute (Mdm)-2 is essential for exercise-induced skeletal muscle angiogenesis. In the current study, we investigated the mechanisms that regulate Mdm2 activity in response to acute exercise and identified VEGF-A as a key stimulator of Mdm2 phosphorylation on Ser(166) (p-Ser166-Mdm2). VEGF-A and p-Ser166-Mdm2 protein levels were measured in human and rodent muscle biopsy specimens after 1 bout of exercise. VEGF-A-dependent Mdm2 phosphorylation was demonstrated in vivo in mice harboring myofiber-specific deletion of VEGF-A (mVEGF(-/-)) and in vitro in primary human and rodent endothelial cells (ECs). Exercise increased VEGF-A and p-Ser166-Mdm2 protein levels respectively by 157 and 68% in human muscle vs. pre-exercise levels. Similar results were observed in exercised rodent muscles compared to sedentary controls; however, exercise-induced Mdm2 phosphorylation was significantly attenuated in mVEGF(-/-) mice. Recombinant VEGF-A elevated p-Ser166-Mdm2 by 50-125% and stimulated migration by 33% in ECs when compared to untreated cells, whereas the Mdm2 antagonist Nutlin-3a abrogated VEGF-driven EC migration. Finally, overexpression of a Ser166-Mdm2 phosphorylation mimetic increased EC migration, increased Mdm2 to FoxO1 binding (+55%), and decreased FoxO1-dependent gene expression compared with ECs overexpressing WT-Mdm2. Our results suggest that VEGF-mediated Mdm2 phosphorylation on Ser(166) is a novel proangiogenic pathway within the skeletal muscle.

Diabetes is rapidly induced in young male Sprague-Dawley rats following treatment with exogenous corticosterone (CORT) and a high-fat diet (HFD). Regular exercise alleviates insulin insensitivity and improves pancreatic β-cell function in insulin-resistant/diabetic rodents, but its effect in an animal model of elevated glucocorticoids is unknown. We examined the effect of voluntary exercise (EX) on diabetes development in CORT-HFD-treated male Sprague-Dawley rats (∼6 wk old). Animals were acclimatized to running wheels for 2 wk, then given a HFD, either wax (placebo) or CORT pellets, and split into 4 groups: placebo-sedentary (SED) or -EX and CORT-SED or -EX. After 2 wk of running combined with treatment, CORT-EX animals had reduced visceral adiposity, and increased skeletal muscle type IIb/x fiber area, oxidative capacity, capillary-to-fiber ratio and insulin sensitivity compared with CORT-SED animals (all P < 0.05). Although CORT-EX animals still had fasting hyperglycemia, these values were significantly improved compared with CORT-SED animals (14.3 ± 1.6 vs. 18.8 ± 0.9 mM). In addition, acute in vivo insulin response to an oral glucose challenge was enhanced ∼2-fold in CORT-EX vs. CORT-SED ( P < 0.05) which was further demonstrated ex vivo in isolated islets. We conclude that voluntary wheel running in rats improves, but does not fully normalize, the metabolic profile and skeletal muscle composition of animals administered CORT and HFD.

Aim The source of vascular endothelial growth factor-A (VEGF-A) may influence vascular function. Exercise-induced vascular growth has been attributed to elevated metabolic demand and to increased blood flow, involving the production of VEGF-A by skeletal muscle and by endothelial cells respectively. We hypothesized that muscle-derived VEGF-A is not required for vascular adaptations to blood flow in skeletal muscle, as this remodelling stimulus originates within the capillary. Methods Myocyte-specific VEGF-A (mVEGF-/-) deleted mice were treated for 7–21 days with the vasodilator prazosin to produce a sustained increase in skeletal muscle blood flow. Results Capillary number increased in the extensor digitorum longus (EDL) muscle in response to prazosin in wild type but not mVEGF-/-mice. Prazosin increased the number of smooth muscle actin-positive blood vessels in the EDL of wild-type but not mVEGF-/- mice. The average size of smooth muscle actin-positive blood vessels also was smaller in knockout mice after prazosin treatment. In response to prazosin treatment, VEGF-A mRNA was elevated within the EDL of wild-type but not mVEGF-/- mice. Ex vivo incubation of wild-type EDL with a nitric oxide donor increased VEGF-A mRNA. Likewise, we demonstrated that nitric oxide donor treatment of cultured myoblasts stimulated an increase in VEGF-A mRNA and protein. Conclusion These results suggest a link through which flow-mediated endothelial-derived signals may promote myocyte production of VEGF-A. In turn, myocyte-derived VEGF-A is required for appropriate flow-mediated microvascular remodelling. This highlights the importance of the local environment and paracrine interactions in the regulation of tissue perfusion.

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.

Type-1 diabetes mellitus (T1D) causes impairments within the skeletal muscle microvasculature. Both regular exercise and prazosin have been shown to improve skeletal muscle capillarization and metabolism in healthy rats through distinct angiogenic mechanisms. The aim of this study was to evaluate the independent and additive effects of voluntary exercise and prazosin treatment on capillary-to-fiber ratio (C:F) in streptozotocin (STZ)-treated diabetic rats. STZ (65 mg/kg) was intraperitoneally administered to male Sprague-Dawley rats ( n = 36) to induce diabetes, with healthy, nondiabetic, sedentary rats ( n = 10) as controls. The STZ-treated rats were then divided into sedentary (SED) or exercising (EX; 24-h access to running wheels) groups and then further subdivided into prazosin (Praz) or water (H2O) treatment groups: nondiabetic-SED-H2O, STZ-SED-H2O, STZ-EX-H2O, STZ-SED-Praz, and STZ-EX-Praz. After 3 wk, untreated diabetes significantly reduced the C:F in tibialis anterior (TA) and soleus muscles in the STZ-SED-H2O animals (both P < 0.05). Voluntary exercise and prazosin treatment independently resulted in a normalization of C:F within the TA (1.86 ± 0.12 and 2.04 ± 0.03 vs 1.71 ± 0.09, P < 0.05) and the soleus (2.36 ± 0.07 and 2.68 ± 0.14 vs 2.13 ± 0.12, P < 0.05). The combined STZ-EX-Praz group resulted in the highest C:F within the TA (2.26 ± 0.07, P < 0.05). Voluntary exercise volume was negatively correlated with fed blood glucose levels ( r2 = −0.7015, P < 0.01) and, when combined with prazosin, caused further enhanced nonfasted glucose ( P < 0.01). Exercise and prazosin reduced circulating nonesterified fatty acids more than either stimulus alone ( P < 0.05). These results suggest that the distinct stimulation of angiogenesis, with both regular exercise and prazosin treatment, causes a cooperative improvement in the microvascular complications associated with T1D. NEW & NOTEWORTHY It is currently well established that poorly controlled diabetes reduces both skeletal muscle mass and muscle capillarization. These muscle-specific features of diabetes may, in turn, compromise insulin sensitivity and glucose control. Using a model of streptozotocin-induced diabetes, we show the vascular complications linked with disease and how chronic exposure to exercise and prazosin (an α1-adrenergic antagonist) can reduce these complications and improve glycemic control.

Background/Aims Impaired regulation of apoptosis has been suggested to play a role in the pathogenesis of Primary Biliary Cirrhosis (PBC). In this study, we analysed a signalling pathway that comprises the transcription factor FoxO3a and its downstream target Bim, a Bcl-2 interacting mediator of apoptosis. Materials & Methods The tissues examined included livers explanted from patients with cirrhotic PBC, primary sclerosing cholangitis (PSC), alcoholic liver disease (ALD) and liver biopsies from patients with non-cirrhotic PBC. Large margin resections of hepatocellular carcinoma were used as controls. Results Expression of FoxO3a and Bim mRNA was significantly enhanced in both non-cirrhotic and end-stage PBC (2.2-fold and 4.3-fold increases, respectively), but not in the other disorders. Similarly, FOXO3a protein level was increased in end-stage PBC (P

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.

Peripheral arterial disease (PAD) is a common vascular disease that reduces blood flow capacity to the legs of patients. PAD leads to exercise intolerance that can progress in severity to greatly limit mobility, and in advanced cases leads to frank ischemia with pain at rest. It is estimated that 12 to 15 million people in the United States are diagnosed with PAD, with a much larger population that is undiagnosed. The presence of PAD predicts a 50% to 1500% increase in morbidity and mortality, depending on severity. Treatment of patients with PAD is limited to modification of cardiovascular disease risk factors, pharmacological intervention, surgery, and exercise therapy. Extended exercise programs that involve walking approximately five times per week, at a significant intensity that requires frequent rest periods, are most significant. Preclinical studies and virtually all clinical trials demonstrate the benefits of exercise therapy, including improved walking tolerance, modified inflammatory/hemostatic markers, enhanced vasoresponsiveness, adaptations within the limb (angiogenesis, arteriogenesis, and mitochondrial synthesis) that enhance oxygen delivery and metabolic responses, potentially delayed progression of the disease, enhanced quality of life indices, and extended longevity. A synthesis is provided as to how these adaptations can develop in the context of our current state of knowledge and events known to be orchestrated by exercise. The benefits are so compelling that exercise prescription should be an essential option presented to patients with PAD in the absence of contraindications. Obviously, selecting for a lifestyle pattern that includes enhanced physical activity prior to the advance of PAD limitations is the most desirable and beneficial.

The matrix metalloproteinases are a family of matrix degrading enzymes that are important regulators of extracellular matrix remodelling and cellular function. This article presents a review of the mechanisms through which MMPs are regulated including transcriptional, post-transcriptional and post-translational events. Regulation of MMPs within the cardiovascular system and their contribution to physiological and pathological events will be discussed.

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.

Elevated shear stress within the skeletal muscle microvasculature is implicated in the induction of a longitudinal splitting form of angiogenesis, which is characterized by the lack of basement membrane breakage. We investigated whether the transcriptional regulator, Ets-1, is responsive to changes in hemodynamic forces and if so, whether Ets-1 controls microvascular endothelial cell integrity by inducing the expression of inhibitors of matrix degrading proteases. Rats were treated with prazosin for 2, 4 and 7 days to increase in microvascular shear stress in hindlimb skeletal muscles. In complimentary in vitro experiments, rat microvascular skeletal muscle endothelial cells were exposed to laminar shear stress (15 dyne/cm2) for 0.5, 2, and 24 hours. TaqMan PCR analysis of laser microdissected capillaries isolated from EDL muscles demonstrated transient (after 2 days) induction of Ets-1 gene expression. In cultured cells, a transient up-regulation of Ets-1 mRNA was observed after 2hr shear stimulation, accompanied by increased phosphorylation of Ets-1 and enhanced Ets-1 DNA binding activity. This response was modulated by ERK1/2 and p38 MAP kinases, but was not dependent on NOS or COX-2 activity. PAI-1, TIMP-1 and TIMP-3 mRNA were elevated significantly in prazosin treated EDL, and in response to shear stimulation in vitro. In cultured endothelial cells, Ets-1 RNA interference abolished the shear-induced increases in Ets-1, PAI-1, TIMP-1 and TIMP-3 mRNA expression. These results suggest that enhanced laminar shear stress may act to preserve the integrity of microvascular walls in part through Ets-1-dependent induction of protease inhibitors.

Proteolysis of the basement membrane and interstitial matrix occurs early in the angiogenic process and requires matrix metalloproteinase (MMP) activity. Skeletal muscle microvascular endothelial cells exhibit robust actin stress fibers, low levels of membrane type 1 (MT1)-MMP expression, and minimal MMP-2 activation. Depolymerization of the actin cytoskeleton increases MT1-MMP expression and MMP-2 activation. Rho family GTPases are regulators of actin cytoskeleton dynamics, and their activity can be modulated in response to angiogenic stimuli such as vascular endothelial growth factor (VEGF). Therefore, we investigated their roles in MMP-2 and MT1-MMP production. Endothelial cells treated with H1152 [an inhibitor of Rho kinase (ROCK)] induced stress fiber depolymerization and an increase in cortical actin. Both MMP-2 and MT1-MMP mRNA increased, which translated into greater MMP-2 protein production and activation. ROCK inhibition rapidly increased cell surface localization of MT1-MMP and increased PI3K activity, which was required for MMP-2 activation. Constitutively active Cdc42 increased cortical actin polymerization, phosphatidylinositol 3-kinase activity, MT1-MMP cell surface localization, and MMP-2 activation similarly to inhibition of ROCK. Activation of Cdc42 was sufficient to decrease RhoA activity. Capillary sprout formation in a three-dimensional collagen matrix was increased in cultures treated with RhoAN19 or Cdc42QL and, conversely, decreased in cultures treated with dominant negative Cdc42N17. VEGF stimulation also induced activation of Cdc42 while inhibiting RhoA activity. Furthermore, VEGF-dependent activation of MMP-2 was reduced by inhibition of Cdc42. These results suggest that Cdc42 and RhoA have opposing roles in regulating cell surface localization of MT1-MMP and MMP-2 activation.

Persistent elevation in shear stress within conduit or resistance arteries causes structural luminal expansion, which serves to normalize shear stress while maintaining increased flow to the downstream vasculature. Although it is known that this adaptation involves cellular proliferation and remodeling of the extracellular matrix, the specific cellular events underlying these responses are poorly understood. Matrix metalloproteinases (MMPs) contribute to extensive remodeling of the extracellular matrix in conduit vessels and vein grafts exposed to high flow. However, involvement of MMPs in remodeling of small muscular collateral arteries, which are exposed to less severe increases in shear stress, has not been tested. We utilized an established model of outward remodeling in mesenteric collateral arteries to determine whether MMPs were upregulated during the remodeling response and to test whether MMP activity was required for luminal expansion. By 4 days, MMP-2 and membrane type 1 MMP (MT1-MMP), but not MMP-9, protein levels were significantly elevated in collateral arteries, as assessed by gelatin zymography and immunostaining. MMP-2 and MT1-MMP proteins, together with their respective transcriptional activators c-Jun and Egr-1 were localized predominantly to the smooth muscle layer of the collateral arteries. The general MMP inhibitor doxycycline prevented luminal expansion of collateral arteries but did not affect the endothelial cell proliferative or medial growth responses. In conclusion, this study provides evidence that MMP-2 and MT1-MMP are upregulated in collateral arteries exposed to elevated shear stress and that MMP activity is essential for the full remodeling response that leads to outward luminal expansion.

Angiogenesis, which is essential for the physiological adaptation of skeletal muscle to exercise, occurs in response to the mechanical forces of elevated capillary shear stress and cell stretch. Increased production of VEGF is a characteristic of endothelial cells undergoing either stretch- or shear-stress-induced angiogenesis. Because VEGF production is regulated by hypoxia inducible factors (HIFs), we examined whether HIFs play a significant role in the angiogenic process initiated by these mechanical forces. Rat extensor digitorum longus (EDL) muscles were overloaded to induce stretch, or exposed to the dilator prazosin to elevate capillary shear stress, and capillaries from these muscles were isolated by laser capture microdissection for RNA analysis. HIF-1α and HIF-2α transcript levels increased after 4 and 7 days of stretch, whereas a transient early induction of HIF-1α and HIF-2α transcripts was detected in capillaries from prazosin-treated muscles. Skeletal muscle microvascular endothelial cells exposed to 10% stretch in vitro showed an elevation in HIF-1α and HIF-2α mRNA, which was preceded by increases in HIF-binding activity. Conversely, HIF-1α and HIF-2α mRNA were reduced significantly, and HIF-α proteins were undetectable, after 24 h exposure to elevated shear stress (16 dyn cm−2 (16 ×10−5 N cm−2). Given the disparate regulation of HIFs in response to these mechanical stimuli, we tested the requirement of HIF-α proteins in stretch- and shear-stress-induced angiogenesis by impeding HIF accumulation through use of the geldanamycin derivative 17-DMAG. Treatment with 17-DMAG significantly impaired stretch-induced, but not shear-stress-induced, angiogenesis. Together, these results illustrate that activation of HIF-1α and HIF-2α contributes significantly to stretch- but not to shear-stress-induced capillary growth.

Capillaries, which are the smallest and most abundant type of blood vessel, form the primary site of gas, nutrient, and waste transfer between the vascular and tissue compartments. Skeletal muscle exhibits the capacity to generate new capillaries (angiogenesis) as an adaptation to exercise training, thus ensuring that the heightened metabolic demand of the active muscle is matched by an improved capacity for distribution of gases, nutrients, and waste products. This review summarizes the current understanding of the regulation of skeletal muscle capillary growth. The multi-step process of angiogenesis is coordinated through the integration of a diverse array of signals associated with hypoxic, metabolic, hemodynamic, and mechanical stresses within the active muscle. The contributions of metabolic and mechanical factors to the modulation of key pro- and anti-angiogenic molecules are discussed within the context of responses to a single aerobic exercise bout and short-term and long-term training. Finally, the paradoxical lack of angiogenesis in peripheral artery disease and diabetes and the implications for disease progression and muscle health are discussed. Future studies that emphasize an integrated analysis of the mechanisms that control skeletal muscle capillary growth will enable development of targeted exercise programs that effectively promote angiogenesis in healthy individuals and in patient populations.

Adipose tissue (AT) homeostasis and growth are dependent on microvasculature. This capillary network has a large remodeling capacity, a process called angio-adaptation. In response to metabolic alterations linked to obesity, signaling pathways involved in the maintenance of vascular homeostasis of AT appear to be affected. In this context, we studied in C57/Bl6 subject to an high fat diet (HFD) and voluntary exercise protocol of 7weeks, capillarisation of epididymal (eWAT) and subcutaneous adipose tissue (sWAT) by histological marking of CD31, and expression of angiogenic factors: Murinedouble- minute 2 (MDM-2), vascular endothelial growth factor (VEGF-A) and trombospondin (TSP-1). Morphometric analysis of mice showed a significant reduction of 30% in the ratio of AT/total mass in HFD trained mice at 7weeks of exercise. In these mice, % of mass eWAT/total and sWAT/total significantly reduced by 33 and 39%, respectively, which was associated to a significant decrease in adipocytes size by 26% in eWAT and 30% in sWAT of HFD mice at 7weeks of exercise. Biochemical study showed that exercise led to an increase of MDM-2 expression and VEGF-A/TSP-1 ratio and in AT of control and HFD mice after 7weeks of exercise pointing the emerging of a angio-adaptive response in favor of capillary growth in AT, was was confirmed by a significant increase of the capillary density (capillaries/mm 2 ) in AT of control mice (55 and 36% respectively in eWAT and sWAT) and a capillaries/adipocyte ratio significantly increased in HFD trained mice (respectively 16 and 18% in eWAT and sWAT). These results showed for the first time that physical exercise acts as a pro-angiogenic stimulus in AT in favor of capillary growth, thru activation of MDM-2, and VEGF-A/TSP-1 ratio

Prolonged exposure to elevated glucocorticoids (GC) results in skeletal muscle capillary rarefaction, which may promote insulin resistance and limb ischemia. Conversely, treatment with the a1-adren...

Increased production and activation of matrix metalloproteinase-2 (MMP-2) are critical events in skeletal muscle angiogenesis and are known to occur in response to mechanical stresses. We hypothesized that reorganization of the actin cytoskeleton would increase endothelial cell production and activation of MMP-2 and that this increase would require a MAPK-dependent signaling pathway in endothelial cells. The pharmacological actin depolymerization agent cytochalasin D increased expression of MMP-2 and membrane type 1-matrix metalloproteinase (MT1-MMP) mRNA, and this was reduced significantly in the presence of the JNK inhibitor SP600125. Activation of JNK by anisomycin was sufficient to induce expression of both MMP-2 and MT1-MMP mRNA in quiescent cells. Downregulation of c-Jun, a downstream target of JNK, with small interference (si)RNA inhibited MMP-2 expression in response to anisomycin. Inhibition of phosphoinositide 3-kinase (PI3K), but not JNK, significantly decreased the amount of active MMP-2 following cytochalasin D stimulation with a concurrent decrease in MT1-MMP protein. Physiological reorganization of actin occurs during VEGF stimulation. VEGF-induced MMP-2 protein production and activation, as well as MT1-MMP protein production, depended on PI3K activity. VEGF-induced MMP-2 mRNA expression was reduced by inhibition of JNK or by treatment with c-Jun siRNA. In summary, our results provide novel insight into the signaling cascades initiated in the early stages of angiogenesis through the reorganization of the actin cytoskeleton and demonstrate a critical role for JNK in regulating MMP-2 and MT1-MMP mRNA expression, whereas PI3K regulates protein levels of both MMP-2 and MT1-MMP.

Angiogenesis provides a mechanism by which delivery of oxygen and nutrients is adapted to compliment changes in tissue mass or metabolic activity. However, maladaptive angiogenesis is integral to the process of several diseases common in Western countries, including tumor growth, vascular insufficiency, diabetic retinopathy and rheumatoid arthritis. Understanding the process of capillary growth, including the identification and functional analyses of key pro- and anti-angiogenic factors, provides knowledge that can be applied to improve/reverse these pathological states. Initially, angiogenesis research focused predominantly on vascular endothelial growth factor (VEGF) as a main player in the angiogenesis cascade. It is apparent now that participation of multiple angiogenic factors and signal pathways is critical to enable effective growth and maturation of nascent capillaries. The purpose of this review is to focus on recent progress in identifying angiogenesis signaling pathways that show promise as targets for successful induction or inhibition of capillary growth. The strategies applied to achieve these contradictory tasks are discussed within the framework of our existing fundamental knowledge of angiogenesis signaling cascades, with an emphasis on comparing the employment of distinctive tactics in modulation of these pathways. Innovative developments that are presented include: (1) inducing a pleiotropic response via activation or inhibition of angiogenic transcription factors; (2) modulation of nitric oxide tissue concentration; (3) manipulating the kallikrein-kinin system; (4) use of endothelial progenitor cells as a means to either directly contribute to capillary growth or to be used as a vehicle to deliver "suicide genes" to tumor tissue.

Microvascular endothelial cells embedded within three-dimensional (3D) type I collagen matrixes assemble into cellular networks, a process that requires the upregulation of membrane type 1 (MT1) matrix metalloproteinase (MMP) and MMP-2. The purpose of this study was to identify the signaling pathways responsible for the transcriptional activation of MT1-MMP and MMP-2 in endothelial cells in 3D collagen lattices. We hypothesized that the 3D type I collagen induction of MT1-MMP and MMP-2 is mediated by the mitogen-activated protein kinase family of enzymes. Here, we show that 3D type I collagen elicits a persistent increase in ERK1/2 and JNK activation and a decrease in p38 activation. Inhibition of ERK1/2 or JNK disrupted endothelial network formation in 3D type I collagen lattices, whereas inhibition of p38 promoted network formation. mRNA levels of both MT1-MMP and MMP-2 were attenuated by ERK1/2 inhibition but unaffected by either JNK or p38 inhibition. By contrast, expression of constitutively active MEK was sufficient to stimulate MMP-2 production in a monolayer of endothelial cells cultured on type I collagen. These results provide evidence that signaling through both ERK1/2 and JNK regulates endothelial assembly into cellular networks but that the ERK1/2 signaling cascade specifically regulates network formation and the production of both MT1-MMP and MMP-2 genes in response to 3D type I collagen.

The process of sprouting angiogenesis requires that the endothelial cells degrade the basement membrane matrix and migrate into the interstitial matrix. Matrix metalloproteinases are enzymes capable of cleaving numerous extracellular matrix proteins. Increased production and activity of matrix metalloproteinases in any cell type is associated with a more migratory and invasive phenotype. This paper describes results of recent in-vitro studies of the regulation of transcription and activation of MMP-2 and MT1-MMP in endothelial cells, as well as studies that examined roles of matrix metalloproteinases in activity-induced angiogenesis.Key words: proteolysis, extracellular matrix, angiogenesis, mechanotransduction.

Matrix metalloproteinase-2 (MMP-2) plays a critical role in endothelial cells during the processes of angiogenesis and vascular remodeling. Endothelial cell production of MMP-2 is greatly enhanced when cells are cultured within a three-dimensional type I collagen matrix coinciding with the increased invasive and migratory phenotype of the cells. To define the transcriptional regulation of MMP-2 in rat microvascular endothelial cells, we performed promoter-reporter assays with a series of promoter truncations. Activity of the full promoter was significantly greater in cells cultured within three-dimensional type I collagen compared with cells cultured as a monolayer (two-dimensional) on type I collagen. Truncation of the region encompassing base pairs -1562 to -1375 (relative to the start codon) of the MMP-2 promoter resulted in loss of this differential activity of the MMP-2 promoter. Analysis of this region indicated two putative GATA-2 binding domains between -1437 and -1387. Southwestern blot analysis and electrophoretic mobility shift assays confirmed the binding of GATA-2 to this region of the MMP-2 promoter. Overexpression of GATA-2 in COS-7 cells significantly increased the activity of the full-length MMP-2 promoter-luciferase construct. Endothelial cells expressed greater levels of GATA-2 protein in three-dimensional compared with two-dimensional cultures, and activity of the -1437/-1387 region of the MMP-2 promoter was significantly greater in three-dimensional cultured endothelial cells. Together, these results indicate GATA-2 regulation of the MMP-2 promoter in endothelial cells and that the GATA-2 binding domain is sufficient to drive increased activity of the MMP-2 promoter in response to an extracellular matrix stimulus.

Capillary growth in skeletal muscle occurs via the dissimilar processes of abluminal sprouting or longitudinal splitting, which can be initiated by muscle stretch and elevated shear stress, respectively. The distinct morphological hallmarks of these types of capillary growth suggest that discrete sets of angiogenic mediators play a role in each situation. Because proteolysis and proliferation are two key steps associated with capillary growth, we tested whether differences in the regulation of matrix metalloproteinases (MMPs) or VEGF may be associated with the two types of capillary growth. We found significant increases in MMP-2 total protein and percent activation, and membrane type-1 MMP mRNA levels, compared with controls after muscle stretch but not after shear stress stimulation. In contrast, VEGF protein and endothelial cell proliferation increased after either angiogenic stimulus. We observed that MMP-2 regulation occurs independent of VEGF signaling, because VEGF did not induce MMP-2 production or activation in isolated endothelial cells. Our data suggest that the involvement of MMPs in capillary growth is dependent on the nature of the angiogenic stimulus.

Resume Angiogenesis, the growth of new capillaries, enhances the oxygen delivery capacity of an existing vascular network. This adaptation is a well-documented occurrence in exercising skeletal muscle. The purpose of this review is to summarize our current understanding of the various stimuli that are involved in the initiation of capillary growth in skeletal muscle. The roles of humoral and mechanical signals in the cellular regulation of several key an- giogenic players, vascular endothelial cell growth factor and matrix metalloproteinases, will be discussed. Evidence will be presented supporting the existence of angiogenesis pro- cesses that are distinct from the "classically" defined process. Determining how specific angiogenic stimuli can initiate unique patterns of capillary growth will provide insight into the complex task of developing effective pro-angiogenic therapies. La formation de nouveaux capillaires, ou angiogenese, ameliore la capacite de distribution d'oxygene dans un reseau vasculaire deja existant. Cette adaptation est bien etablie quant au muscle squelettique a l'effort. Cette article trace un bilan des connaissances relatives aux divers stimuli impliques dans l'angiogenese intramusculaire. Il y est traite du role des signaux sanguin et mecanique dans la regulation cellulaire de quelques facteurs angiogeniques cles, notamment le facteur de croissance vasculaire endotheliale et les metalloproteinases matricielles. Des etudes scientifiques demontrent la presence de pro

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.

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. TGFβ production and Smad2/3 phosphorylation are increased by shear stress. Inhibition of TGFβ signaling, either by use of the TGFβ 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 TGFβ signaling pathway is essential to maintain TIMP-1 expression during chronic shear stress.