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

Abstract

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.

Figure 1

Involvement of shear stress (SS)-induced vascular endothelial growth factor receptor 2 (VEGFR2) activation in p38 phosphorylation. Cells were sheared at 12 dynes/cm2 in the presence or absence of 10 μM VEGF receptor 2 kinase inhibitor I (VEGFR2i). Western blotting of cell extracts was performed for (A) VEGFR2-Y1214 phosphorylation (fold change normalized to total VEGFR2 levels) (*P<0.05 vs. control (C), #P<0.05 vs. 0.5 hour SS; n=4); (B) phosphorylated p38 (fold change normalized to total p38 levels) following 2 hours (2h) of shear stress (*P<0.05 vs. C, #P<0.05 vs. SS; n=5); and, (C) and 6 hours (6h) of shear stress (*P<0.05 vs. C, #P<0.05 vs. SS; n=3). (D) VEGFR2i efficacy was assessed by VEGFR2 (Y1214) phosphorylation in response to VEGF stimulation and VEGFR2i treatment (*P<0.05 vs. VEGF, n=3). Values are means ±SE.

Figure 2

Vascular endothelial growth factor receptor 2 (VEGFR2)-Y1214 and p38 phosphorylation in response to vascular endothelial growth factor (VEGF) stimulation. Cells were treated with recombinant VEGF (20 ng/ml) for 5, 15 or 30 minutes prior to protein extraction. Western blotting was performed for (A) VEGFR2-Y1214 phosphorylation (fold change normalized to total VEGFR2 levels) (Fisher’s PLSD, *P<0.05 vs. control (C), n=4) and (B) phosphorylated p38 following 30 min VEGF stimulation (fold change normalized to total p38) (*P<0.05 vs. C; n=4). Values are means ±SE.

Figure 3

Effect of long duration shear stress (SS) and nitric oxide synthase (NOS) inhibition on p38 phosphorylation. Cells were sheared for 6 or 24 hours in the presence or absence of the NOS inhibitor, 30 μM Omega N-nitro-L-arginine (LNNA). Western blotting for (A) phosphorylated p38 after 6 hours (6h) (fold change normalized to total p38 levels) (*P<0.05 vs. control (C), $P<0.05 vs. C+LNNA; n=4) (B) phosphorylated p38 after 24 hours (24h) (fold change normalized to total p38) (*P<0.05 vs. C; n=3). Values are means ±SE.

Figure 4

VEGF production in response to shear stress (SS) and SB203580. Cells were sheared for 6 hours (6h) in the presence or absence of p38 inhibitor SB203580. Western blotting for VEGF-A (fold change normalized to β-actin) (*P<0.05 vs. C, #P<0.05 vs. SS; n=3). Values are means ±SE.

Figure 5

The effects of VEGFR2 activation and NOS inhibition on ERK1/2 phosphorylation in response to shear stress (SS). Western blotting of cell extracts was performed for ERK1/2 phosphorylation (fold change normalized to β-actin) on cells (A) sheared in the presence or absence of 10 μM VEGFR2i for 2 hours (2h) ($P<0.05 vs. C+VEGFR2i; n=5) (B) plated in OptiMem (low serum media) and treated with recombinant VEGF (20 ng/ml) for 30 minutes (*p<0.05 vs. C; n=3) (C) sheared in the presence or absence of 30 μM LNNA for 6 hours (6h) (NS; n=3) and (D) following 24 hours (24h) of shear stress (NS; n=3). Values are means ±SE.

Figure 6

p38 phosphorylation in vivo in response to prazosin treatment. Prazosin (Praz), an alpha1-adrenergic receptor inhibitor, was administered to male Sprague-Dawley rats for up to 7 days (7D). Protein extracts from extensor digitorum longus muscles were analyzed for phosphorylated p38 by Western blotting. (A) phosphorylated p38 after 1, 2 or 4 days (4D) Praz (fold change normalized to total p38) (*p<0.05 vs. C, n=4). (B) phosphorylated p38 after 7 days (7D) Praz (fold change normalized to total p38) (NS, n=4). Values are means ±SE.

Figure 7

Effect of prazosin treatment on p38 phosphorylation in the capillaries of the extensor digitorum longus (EDL). Prazosin was administered to male Sprague-Dawley rats for up to 7 days. EDL muscle cross sections were stained with anti-phospho-p38 (red) and counterstained with fluorescein griffonia simplicifolia lectin I (Vector Laboratories) (green) to visualize capillaries. Sections were viewed by confocal microscopy (Olympus Fluoview 300, z-series, 10 slices). (A) pp38 and (B) lectin/phospho-p38 overlay of a muscle section of an untreated rat. (C) p-p38 and (D) lectin/phospho-p38 staining overlay of a muscle section of a 2 day prazosin treated animal. Arrows indicate sites of p38 phosphorylation co-localized to capillaries. (E) p-p38 and (F) lectin/phosphop38 staining overlay of a muscle section of a 4 day prazosin treated rat. (G) p-p38 and (H) lectin/phospho-p38 staining overlay of a muscle section of a 7 day prazosin treated animal.

Figure 8

Capillary morphology and muscle capillarity in response to prazosin and SB203580 treatment. Prazosin (Praz) and the p38 inhibitor SB203580, or its inactive analog SB202474, was administered to male Sprague-Dawley rats for 7 days (D) by mini-osmotic pump (1μg/μl, 0.5 μl/hour). The extensor hallucis proprius (EHP) and extensor digitorum longus (EDL) from each rat was extracted and prepared for electron microscopy or muscle capillarity analysis respectively. Electron microscopy of the EHP revealed (A) a quiescent state of the capillary in the control animal (RBC = red blood cell). (B) prazosin treatment induces luminal projections (arrows) and pronounced vesicles throughout the endothelium. (C) p38 inhibition resembles the control (D) p38 inhibition and prazosin treatment also resemble the control, with absence of luminal projections. The scale bar represents 1 μm, and is equivalent for each panel. (E) Capillary to muscle fibre ratio in the EDL (*P<0.05 vs. control (C)+SB202474, #P<0.05 vs. shear stress (SS)+202474; n=4). Values are means ±SE.