In vivo angiogenesis in adult rat skeletal muscle: early changes in capillary network architecture and ultrastructure

The individual structural stages in capillary growth have been identified during development and under pathological circumstances in adults (wound healing, tumors), but there are no data to indicate whether these steps are similar when angiogenesis is induced in a fully differentiated microvascular bed in normal, uninjured adult skeletal muscle. In this study changes in capillary ultrastructure were correlated with capillary density and network morphology to elucidate the sequelae of angiogenesis in adult rat extensor digitorum longus (EDL) muscle whose activity was increased by stimulation at 10rHz (8rh/day). This resulted in an increased capillary/fiber (C/F) ratio (based on staining for alkaline phosphatase) after 4rdays; by 7rdays C/F ratio was increased further, by approximately 50%. The ultrastructure of capillary endothelium in both the EDL and extensor hallucis proprius (EHP) was similar to control muscles after 2rdays of stimulation, whereas endothelial cells in some capillaries in muscle stimulated for 4rdays revealed signs of metabolic activation such as proliferation of organelles (Golgi apparatus, endoplasmic reticulum, ribosomes and mitochondria) and fewer pinocytic vesicles. Luminal surfaces were often irregular with numerous pseudopodial processes. Basement membranes were always present but amorphous regions were observed, particularly near pericyte processes. Unusually small capillary profiles, with either a slit-like lumen or with cisternae but no lumen, probably represented capillary sprouts. The interstitium contained increased collagenous and granular extracellular matrix surrounding capillaries, and numerous activated fibroblasts which were closely apposed to many capillaries. Capillary growth in EHP was also evaluated by confocal microscopy using whole mounts. The complex pattern of vessels underwent remodelling between 2 and 7rdays of stimulation, resulting in more tortuous capillaries with numerous sprouts and loops. These combined observations suggest that angiogenesis may occur by a combination of sprouting, intussusceptive growth and elongation; also, that activation of endothelial cells occurs at the same time as disturbance of basement membranes during the earliest phase of growth and remodelling of the capillary bed. These changes are postulated to occur in connection with increased shear stress and/or capillary wall tension, which have been demonstrated previously.     

Red blood cells increase secretion of matrix metalloproteinases from human lung fibroblasts in vitro

Tissue remodeling is an important process in many inflammatory and fibrotic lung disorders. RBC may in these conditions interact with extracellular matrix (ECM). Fibroblasts can produce and secrete matrix components, matrix-degrading enzymes (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). Imbalance in matrix synthesis/degradation may result in rearrangement of tissue architecture and lead to diseases such as emphysema or fibrosis. Neutrophil elastase (NE), a protease released by neutrophils, is known to activate MMP. We hypothesized that RBC can stimulate secretion of MMPs from human lung fibroblasts and that NE can augment this effect. Human fetal lung fibroblasts were cultured in floating collagen gels with or without RBC. After 4 days, the culture medium was analyzed with gelatin zymography, Western blot, and ELISA for MMP-1, -2, -3 and TIMP-1, -2. RBC augmented NE-induced fibroblast-mediated collagen gel contraction compared with NE alone (18.4+/-1.6%, 23.7+/-1.4% of initial gel area, respectively). A pan-MMP inhibitor (GM-6001) completely abolished the stimulating effect of NE. Gelatin zymography showed that RBC stimulated MMP-2 activity and that NE enhanced conversion to the active form. Addition of GM-6001 completely inhibited MMP-2 activity in controls, whereas it only partially altered RBC-induced MMP activity. Western blot confirmed the presence of MMP-1 and MMP-3 in fibroblasts stimulated with RBC, and ELISA confirmed increased concentrations of pro-MMP-1. We conclude that stimulation of MMP secretion by fibroblasts may explain the ability of RBC to augment fibroblast-mediated collagen gel contraction. This might be a potential mechanism by which hemorrhage in inflammatory conditions leads to ECM remodeling.     

Matrix metalloproteinase inhibitor GM 6001 attenuates keratinocyte migration, contraction and myofibroblast formation in skin wounds

In this study, we examined the impact of matrix metalloproteinases (MMP) on epithelialization, granulation tissue development, wound contraction, and alpha-smooth muscle actin (ASMA) expression during cutaneous wound repair through systemic administration of the synthetic broad-spectrum MMP inhibitor GM 6001 (N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide). Four full-thickness excisional wounds (50 mm2) on the back of 22 young female Sprague-Dawley rats, 12 treated with GM 6001 100 mg/kg and 10 with vehicle, were allowed to heal by secondary intention. GM 6001-treated wounds were minimally resurfaced with neoepithelium, despite unaltered keratinocyte proliferation in wound edges, whereas control wounds were completely covered with 3-7 cell layers of parakeratinized epithelium on post-wounding day 7. Hydroxyproline concentration, a marker of collagen, and cell proliferation in granulation tissue did not differ significantly between GM 6001-treated and control groups. Impaired wound contraction (P < 0.01) was associated with a dramatic reduction of ASMA-positive myofibroblasts in granulation tissue of GM 6001 wounds. This was not due to GM6001 blocking transforming growth factor-beta1 (TGF-beta1)-induced myofibroblast differentiation since GM 6001 did not inhibit TGF-beta1-induced ASMA expression and force generation in cultured rat dermal fibroblasts. The profound impairment of skin repair by the nonselective MMP inhibitor GM 6001 suggests that keratinocyte resurfacing, wound contraction, and granulation tissue organization are highly MMP-dependent processes.     

Muscle capillaries in tenotomized human muscles

The capillaries were studied in tenotonized human muscles after tenotomy or spontaneous rupture of the tendon. The mean capillary area was not significantly different in the patients with tenotomy, or with spontaneous rupture of the tendon, as compared to intact muscles. The mean basement membrane area as a percentage of total capillary area was significantly larger and the size capillary lumen significantly smaller in the injured than in the intact muscles. The alterations of the capillaries were time-dependent.     

Morphometric evaluation of capillary basement membrane thickness in the quadriceps muscle of diabetic and nondiabetic Chinese hamsters

Quadriceps muscle capillaries from 19-23 month old genetically diabetic (XA and AC) and nondiabetic (M) subline Chinese hamsters were morphometrically evaluated to determine if capillary basement membrane thickening (CBMT) is a quantifiable complication of diabetes. Significant CBMT was present in the diabetic XA Chinese hamsters (49.37 nm +/- 17.81, p less than 0.007) in comparison with the nondiabetic M hamsters (34.08 nm +/- 9.98). Although there was a trend towards expansion of the muscle capillary basement membranes in the diabetic AC Chinese hamsters, the value was not statistically significant. A nested analysis of variance showed that the greatest source of variation in basement membrane thickness occurred among capillaries within each animal. In addition, a positive correlation (r = 0.62; p less than 0.002) existed between blood glucose levels and CBMT in the XA subline. These data should serve as guidelines for evaluation of antimicrovascular disease compounds which will be tested to determine if they prevent or retard microangiopathy in the diabetic Chinese hamster.     

Effects of aging on capillary geometry and hemodynamics in rat spinotrapezius muscle

The effects of aging on muscle microvascular structure and function may play a key role in performance deficits and impairment of O2 exchange within skeletal muscle of senescent individuals. To determine the effects of aging on capillary geometry, red blood cell (RBC) hemodynamics, and hematocrit in a muscle of mixed fiber type, spinotrapezius muscles from Fischer 344 x Brown Norway hybrid rats aged 6-8 mo [young (Y); body mass 421 +/- 10 g, n = 6] and 26-28 mo [old (O); 561 +/- 12 g, n = 6] were observed by high-resolution transmission light microscopy under resting conditions. The percentage of RBC-perfused capillaries (Y: 78 +/- 3%; O: 75 +/- 2%) and degree of tortuosity and branching (Y: 13 +/- 2%; O: 13 +/- 2%, additional capillary length) were not different in O vs. Y muscles. Lineal density of RBC-perfused capillaries in O was significantly reduced (Y: 30.7 +/- 1.8, O: 22.8 +/- 3.1 capillaries/mm; P < 0.05). However, RBC-perfused capillaries from O rats (n = 78) exhibited increased RBC velocity (VRBC) (Y: 219 +/- 12, O: 310 +/- 14 microm/s; P < 0.05) and RBC flux (FRBC) (Y: 27 +/- 2, O: 41 +/- 2 RBC/s; P < 0.05) vs. Y rats (n = 66). Thus O2 delivery per unit of muscle was not different between groups (Y: 894 +/- 111, O: 887 +/- 118 RBC. s-1. mm muscle-1). Capillary hematocrit was not different in Y vs. O rats (Y: 26 +/- 1%, O: 28 +/- 1%: P > 0.05). These data indicate that in resting spinotrapezius muscle, aging decreases the lineal density of RBC-perfused capillaries while increasing mean VRBC and FRBC within those capillaries. Whereas muscle conductive O2 delivery and capillary hematocrit were unchanged, elevated VRBC reduces capillary RBC transit time and may impair the diffusive transport of O2 from blood to myocyte particularly under exercise conditions.     

Internal division of capillaries in rat skeletal muscle in response to chronic vasodilator treatment with α1-antagonist prazosin

Chronic vasodilatation represents a stimulus for capillary growth associated with increased luminal shear stress. We have examined the ultrastructure of more than 2000 capillaries to establish whether the sequence of angiogenesis in response to this stimulus is similar to that described during development and under pathological circumstances. Administration of the α₁-blocker prazosin to rats for 2 weeks led to a greater capillary length density in extensor hallucis proprius muscles without any change in capillary tortuosity: J _v(c,f)=262±54 compared with 350±17 mm^–2, control compared with prazosin (P<0.002). There were obvious signs of endothelial cell (EC) activation after prazosin treatment, including an increased proportion of capillaries with rough endoplasmic reticulum, large cytoplasmic vacuoles, thickened endothelium and an irregular luminal surface. Capillaries from control muscles had a maximum of three ECs in cross section, whereas four ECs were noted in 0.8+0.5% of capillaries after 1 week (n.s.) and 2.5±0.9% after 2 weeks (P<0.01) of treatment. This could be due to elongation and/or migration of ECs, as cell proliferation has not been described at these time points. There was also an increase in the proportion of capillaries having a narrow, slit-like lumen (1.7±0.8% of controls; 7.1±1.9% at 1 week; 8.8±2.5% at 2 weeks; P<0.02), some of which were smaller in size (less than 2 μm diameter) than in controls (3–5 μm) and/or “seamless”, i.e. lacking EC junctions. These may represent newly formed vessels. Focal discontinuity of the basement membrane and abluminal EC processes were rarely seen, and capillary growth by abluminal sprouting appeared to be very infrequent (less than 0.001% of profiles). Of more importance was growth starting from the luminal side. Significantly more thin cytoplasmic processes were observed protruding into the lumen of capillaries after 1 week (47.5±6.2%, P<0.001) and 2 weeks of prazosin (34.2±5.5%, P<0.05) than in control vessels (16.7±3.9%). Some of these traversed the entire lumen and connected with endothelium of the opposite side, probably involving membrane fusion, resulting in the appearance of a double lumen. Individual capillaries with a complete double lumen were observed after 2 weeks’ prazosin but comparatively rarely, in only four out of six muscles. These findings indicate a pattern of luminal growth which is completely different from intussusceptive growth previously described during development, and from the abluminal capillary sprouting seen under pathological circumstances.     

Endothelial NOS is main mediator for shear stress-dependent angiogenesis in skeletal muscle after prazosin administration

The increase of wall shear stress in capillaries by oral administration of the alpha1-adrenergic receptor antagonist prazosin induces angiogenesis in skeletal muscles. Because endothelial nitric oxide synthase (eNOS) is upregulated in response to elevated wall shear stress, we investigated the relevance of eNOS for prazosin-induced angiogenesis in skeletal muscles. Prazosin and/or the NOS inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) were given to C57BL/6 wild-type mice and eNOS-knockout mice for 14 days. The capillary-to-fiber (C/F) ratio and capillary density (CD; no. of capillaries/mm2) were determined in frozen sections from extensor digitorum longus (EDL) muscles of these mice. Immunoblotting was performed to quantify eNOS expression in endothelial cells isolated from skeletal muscles, whereas VEGF (after precipitation with heparin-agarose) and neuronal NOS (nNOS) concentrations were determined in EDL solubilizates. In EDL muscles of C57BL/6 mice treated for 14 days, the C/F ratio was 28% higher after prazosin administration and 11% higher after prazosin and L-NAME feeding, whereas the CD increased by 21 and 13%, respectively. The C/F ratio was highest after day 4 of prazosin treatment and decreased gradually to almost constant values after day 8. Prazosin administration led to elevation of eNOS expression. VEGF levels were lowest at day 4, whereas nNOS values decreased after day 8. In EDL muscles of eNOS-knockout mice, no significant changes in C/F ratio, CD, or VEGF and nNOS expression were observed in response to prazosin administration. Our data suggest that the presence of eNOS is essential for prazosin-induced angiogenesis in skeletal muscle, albeit other signaling molecules might partially compensate for or contribute to this angiogenic activity. Furthermore, subsequent remodeling of the capillary system accompanied by sequential downregulation of VEGF and nNOS in skeletal muscle fibers characterizes shear stress-dependent angiogenesis.     

Early glomerular alterations in genetically determined low nephron number

An association between low nephron number and subsequent development of hypertension in later life has been demonstrated. The underlying pathomechanisms are unknown, but glomerular and postglomerular changes have been discussed. We investigated whether such changes are already present in prehypertensive "glial cell line-derived neurotrophic growth factor" heterozygous mice (GDNF+/-) with lower nephron number. Twenty-six-week-old mice [22 GDNF+/-, 29 C57B6 wild-type control (wt)] were used for in vivo experiments with intra-arterial and tail cuff blood pressure measurements. After perfusion fixation, kidneys were investigated with morphological, morphometric, stereological, and immunohistochemical techniques and TaqMan PCR analysis. As expected at this age, blood pressure was comparable between GDNF+/- and wt. Nephron number per kidney was significantly lower in GDNF+/- than in wt (-32.8%, P < 0.005), and mean glomerular volume was significantly higher (+49.5%, P < 0.001). Renal damage scores, glomerular and tubular proliferation, analysis of intrarenal arteries and peritubular capillaries, expression of relevant tubular transporter proteins, as well as gene expression of profibrotic, proinflammatory, or prohypertensive markers were not significantly different between GDNF+/- and wt. Compensatory glomerular hypertrophy in GDNF+/- was accompanied by higher numbers of endothelial and mesangial cells as well as PCNA-positive glomerular cells, whereas podocyte density was significantly reduced. Further electron microscopic analysis showed marked thickening of glomerular basement membrane. In conclusion, lower nephron number is associated with marked early glomerular structural changes, in particular lower capillary supply, reduced podocyte density, and thickened glomerular basement membrane, that may predispose to glomerular sclerosis.     

Cellular changes in normal blood capillaries undergoing regression after inhibition of VEGF signaling

The vasculature of the embryo requires vascular endothelial growth factor (VEGF) during development, but most adult blood vessels lose VEGF dependence. However, some capillaries in the respiratory tract and selected other organs of adult mice regress after VEGF inhibition. The present study sought to identify the sequence of events and the fate of endothelial cells, pericytes, and vascular basement membrane during capillary regression in mouse tracheas after VEGF signaling was blocked with a VEGF-receptor tyrosine kinase inhibitor AG-013736 or soluble receptor construct (VEGF Trap or soluble adenoviral VEGFR-1). Within 1 day, patency was lost and fibrin accumulated in some tracheal capillaries. Apoptotic endothelial cells marked by activated caspase-3 were present in capillaries without blood flow. VEGF inhibition was accompanied by a 19% decrease in tracheal capillaries over 7 days and 30% over 21 days. During this period, desmin/NG2-immunoreactive pericytes moved away from regressing capillaries onto surviving vessels. Empty sleeves of basement membrane, left behind by regressing endothelial cells, persisted for about 2 wk and served as a scaffold for vascular regrowth after treatment ended. The amount of regrowth was limited by the number of surviving basement membrane sleeves. These findings demonstrate that, after inhibition of VEGF signaling, some normal capillaries regress in a systematic sequence of events initiated by a cessation of blood flow and followed by apoptosis of endothelial cells, migration of pericytes away from regressing vessels, and formation of empty basement membrane sleeves that can facilitate capillary regrowth.     

The ultrastructure of the renal corpuscle of a lizard

The few and small renal corpuscles of the lizard Podarcis (= Lacerta) taurica are composed of a tuft of three to four capillaries (glomerulus), Bowman's capsule and mesangium. The thin interdigitated capillary endothelial cells are, in most regions, in contact with the mesangium. In some regions, however, they rest on a bilaminate basement membrane with an electron-dense lamina densa and a less dense lamina rara. Bowman's capsule is composed of visceral and parietal layers. The epithelial cells (podocytes) of the visceral layer bear trabeculae connected to pedicels with microvilli. The pedicels rest on a bilaminate basement membrane which in some regions has a double-layered densa with connecting bands. Generally, this basement membrane is thicker than that of the capillary endothelial cells. The mesangium is composed mostly of irregular satellite cells with large nuclei and cytoplasmic processes, but also has smaller cells with kidney-shaped nuclei and cytoplasmic processes containing microfilaments. The mesangium cells are embedded in a collagenous matrix which extends to invade the area between the epithelial basement membrane and the capillary endothelium. These observations are discussed in relation to the structure and function of vertebrate renal corpuscles with special reference to the mesangium.     

The microcirculatory bed of the normal rat thyroid and during hypokinesia based on scanning electron microscopy data on corrosion preparations

In 12 mature white rats with body mass of 180-230 g, that are kept in tight pencil-cases for 10, 30 and 90 days (3 rats make the control), by means of scanning electron microscopy of corrosive preparations reorganizations of three-dimensional spatial organization of the thyroid microcirculatory bed are studied. In intact animals single-layered network of the perifollicular capillaries consists of widely anastomotic blood microvessels and makes 68 +/- 7% of the follicular surface. At hypokinesia (Hk) lasting for 10 days, the perifollicular capillaries are sharply dilated, the capillary network area makes 74 +/- 4%. A large amount of processes appear on the capillary walls. On the 30th day of Hk unequal distribution of capillaries on the follicular surface is noted, that is heterogeneity in organization of the perifollicular capillary networks is manifested. In 90 days of Hk reduction of the capillaries is recorded, rarefied pericapillary network prevails, twistedness of the capillaries is clearly manifested, their complex branching decreases. The capillary network area makes 54 +/- 3% of the follicular area. A large amount of pin-shaped protrusions of the capillary wall appear.     

Hyaluronic acid as an anti-angiogenic shield in the preovulatory rat follicle

Angiogenesis in the preovulatory follicle is confined to the theca cell layers, and penetration of capillaries through the basement membrane into the granulosa cell layers does not occur until after ovulation. However, elevated expression of the angiogenic growth factor (VEGF) has been reported in the cumulus cells surrounding the oocyte, which are expelled from the follicle during ovulation. This spatial and temporal discrepancy between VEGF expression and angiogenesis was studied here in the rat ovarian follicle, and we showed that cumulus cells secrete to the follicular fluid, in addition to VEGF, material with antiangiogenic activity that blocks endothelial cell proliferation, migration, and capillary formation in vitro. Hyaluronic acid produced by the cumulus cells can account for this antiangiogenic activity. Degradation of hyaluronic acid by hyaluronidase restored proliferation and migration of endothelial cells directed toward the cumulus. Inhibition of hyaluronic acid synthesis with 6-diazo-5-oxo-1-norleucine restored endothelial proliferation and migration in vitro, and it also resulted in early penetration of capillaries across the follicular basement membrane in vivo. These results support the role of hyaluronic acid produced by the cumulus cells as a high-molecular-weight, antiangiogenic shield that prevents premature vascularization of the preovulatory follicle by blocking endothelial cell migration and proliferation.     

Enzymatic differentiation of arterial and venous segments of the capillary bed during the development of free muscle grafts in the rat

The revascularization of freely grafted muscles in the rat was studied by histochemical reactions that on frozen sections stain the arterial part of the capillary bed blue (alkaline phosphatase [AP] reaction) and the venous part of the capillary bed red (dipeptidyl peptidase IV [DPP IV] reaction). In 112 rats the extensor digitorum longus or soleus muscles were freely grafted and removed at various times up to 93 days following the surgery. In cross section, the capillaries of a normal muscle show a mosaic pattern of staining for the activity of the two enzymes. After grafting, DPP IV activity of capillaries is lost throughout the entire graft within a day or two; but within ischemic muscle, weak and diffuse AP staining persists in capillary remnants for up to 6 days. In the very periphery of the graft AP staining is also preserved in partially damaged capillaries. By 4 days, new AP-positive capillaries can be identified at the periphery of the graft, and in succeeding days AP-positive capillaries are found progressively nearer the center of the graft. At 7 days, the capillary/muscle-fiber ratios are 66% of normal in the periphery of the graft and 44% in the intermediate zone. DPP IV-stained capillaries are not seen until 7 days after grafting. By 60 days, when the grafts have become stabilized, the mosaic pattern of capillary staining has become reestablished. In mature grafts, the number of capillaries per unit area was slightly higher than that in control muscles, but the capillary/muscle-fiber ratio was slightly lower, owing to the smaller than normal cross-sectional areas of the regenerated muscle fibers.     

The role of laminin-1 in the modulation of radiation damage in endothelial cells and differentiation.

Microvascular dysfunction due to endothelial damage is often associated with the ionizing radiation used during cancer therapy. This radiation-induced capillary injury is a major factor in the inhibition of new vessel growth (angiogenesis) and in disease states such as radiation-induced pneumonitis and nephropathy. Many studies have examined the effects of radiation on endothelial cell function; however, little is known regarding the role the basement membrane plays in radiation-induced endothelial cell damage and angiogenesis. Therefore, we examined the effects of gamma radiation on aortic explants, and in vitro on three endothelial cell types (of artery, vein and capillary origin) irradiated with or without the basement membrane glycoprotein laminin-1. As expected, irradiation inhibited angiogenic sprouting of the aortic explants, endothelial cell proliferation, attachment, migration and differentiation in vitro in a dose-dependent manner. However, the effect of radiation on several of these processes in angiogenesis was reduced when the cells were irradiated on laminin-1. To further evaluate the effects of radiation on endothelial cells, we examined the expression of the vascular endothelial cell growth factor (VEGF) kinase domain region receptor in endothelial cells irradiated in the presence and absence of laminin-1. In endothelial cells irradiated on laminin-1, KDR expression increased 2.5-fold over control levels. Therefore, although radiation has a dose-dependent inhibitory effect on processes associated with angiogenesis in vitro, the presence of the basement membrane glycoprotein laminin-1 during irradiation decreases these effects.     

Short-term reversibility of ultrastructural changes in pulmonary capillaries caused by stress failure.

We previously showed that when the pulmonary capillaries in anesthetized rabbits are exposed to a transmural pressure (Ptm) of approximately 40 mmHg, stress failure of the walls occurs with disruption of the capillary endothelium, alveolar epithelium, or sometimes all layers. The present study was designed to determine whether some of the ultrastructural changes are rapidly reversible when the capillary pressure is reduced. To test this, the Ptm was raised to 52.5 cmH2O for 1 min of blood perfusion and then reduced to 12.5 cmH2O for 3 min of saline-dextran perfusion, followed by intravascular fixation at the same pressure. In another group of animals, the pressure was elevated for 1 min of blood and 3 min of saline-dextran before being reduced. The results were compared with previous studies in which the capillary pressures were maintained elevated at 52.5 cmH2O during the entire procedure. Control studies were also done at sustained low pressures. The results showed that the number of endothelial and epithelial breaks per millimeter and the total fraction area of the breaks were reduced when the pressure was lowered. For example, the number of endothelial breaks per millimeter decreased from 7.1 +/- 2.1 to 2.4 +/- 0.7, and the number of epithelial breaks per millimeter fell from 11.4 +/- 3.7 to 3.4 +/- 0.7. There was evidence that the breaks that closed were those that were initially small and were associated with an intact basement membrane. The results suggest that cells can move along their underlying matrix by rapid disengagement and reattachment of cell adhesion molecules, causing breaks to open or close within minutes.(ABSTRACT TRUNCATED AT 250 WORDS)     

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/cm², 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. J. Cell. Physiol. 222:120–126, 2010. © 2009 Wiley‐Liss, Inc.     

Differential involvement of MMP-2 and VEGF during muscle stretch- versus shear stress-induced angiogenesis

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.