Angiotensin II Evokes Angiogenic Signals within Skeletal Muscle through Co-ordinated Effects on Skeletal Myocytes and Endothelial Cells

Skeletal muscle overload induces the expression of angiogenic factors such as vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-2, leading to new capillary growth. We found that the overload-induced increase in angiogenesis, as well as increases in VEGF, MMP-2 and MT1-MMP transcripts were abrogated in muscle VEGF KO mice, highlighting the critical role of myocyte-derived VEGF in controlling this process. The upstream mediators that contribute to overload-induced expression of VEGF have yet to be ascertained. We found that muscle overload increased angiotensinogen expression, a precursor of angiotensin (Ang) II, and that Ang II signaling played an important role in basal VEGF production in C2C12 cells. Furthermore, matrix-bound VEGF released from myoblasts induced the activation of endothelial cells, as evidenced by elevated endothelial cell phospho-p38 levels. We also found that exogenous Ang II elevates VEGF expression, as well as MMP-2 transcript levels in C2C12 myotubes. Interestingly, these responses also were observed in skeletal muscle endothelial cells in response to Ang II treatment, indicating that these cells also can respond directly to the stimulus. The involvement of Ang II in muscle overload-induced angiogenesis was assessed. We found that blockade of AT1R-dependent Ang II signaling using losartan did not attenuate capillary growth. Surprisingly, increased levels of VEGF protein were detected in overloaded muscle from losartan-treated rats. Similarly, we observed elevated VEGF production in cultured endothelial cells treated with losartan alone or in combination with Ang II. These studies conclusively establish the requirement for muscle derived VEGF in overload-induced angiogenesis and highlight a role for Ang II in basal VEGF production in skeletal muscle. However, while Ang II signaling is activated following overload and plays a role in muscle VEGF production, inhibition of this pathway is not sufficient to halt overload-induced angiogenesis, indicating that AT1-independent signals maintain VEGF production in losartan-treated muscle

Effect of Ang II stimulation on VEGF and MMP-2 expression in C2C12 myotubes.

<p>C2C12 myoblasts were differentiated in 2% serum media for 5–8 days until myotubes had formed. Following myotube formation, cells were switched to Opti-mem reduced serum media and stimulated with Ang II. VEGF mRNA levels were determined by qPCR in response to Ang II (0.1 µM) stimulation for 2 hours (n = 7 per condition) (A). Representative blot and quantification of VEGF protein expression after overnight stimulation with Ang II (0.1 µM) and normalized to tubulin (n = 7 per condition) (B). MMP-2 mRNA levels were assessed in response to 2 hour Ang II (0.1 µM) treatment by qPCR (n = 6 per condition) (C). C2C12 myoblasts were stimuated for either 10 or 30 minutes with 1 µM Ang II and then lysed for protein analysis. P-ERK1/2 (n = 3 per condition) (D) and P-Akt (n = 5 per condition) (E) levels were assessed by Western blotting with activated levels normalized to ß-actin and total Akt levels respectively. Values are presented as mean ± SEM. Student’s t-test and one way ANOVA followed by Bonferroni’s multiple comparison test were used to assess statistical significance which was set as p<0.05. In panels A–D, *denotes p<0.05 vs. untreated cells. C – Control, 10–10 minutes and 30–30 minutes.</p

Muscle overload-induced angiogenesis is attenuated in muscle VEGF KO animals.

<p>Representative inverted grey-scale images of iso-lectin staining (A) were used to calculate the capillary to muscle fiber ratio (n = 10 for WT Sham, n = 4 for KO Sham, n = 7 for WT OL and n = 5 for KO OL) (B). Muscle subjected to overload for 7 days was lysed for qPCR analysis of VEGF (C), MMP-2 (D) and MT1-MMP (E) transcript levels (n = 4 for WT Sham, KO Sham and KO OL and n = 3 for WT OL). Values are presented as mean ± SEM. There was a significant main effect of overload on the capillary to fiber ratio as well as VEGF, MMP-2 and MT1-MMP transcript levels, as assessed by two way ANOVA (*). Bonferroni’s multiple comparison posthoc tests revealed a significant difference between the capillary to fiber ratio of KO sham animals compared WT sham animals (**) and a significant difference in the capillary to fiber ratio and VEGF, MMP-2 and MT1-MMP transcript levels in KO overload animals compared to WT overload animals (#). WT – wildtype, KO – knockout and OL – overload.</p

Effect of Ang II stimulation on VEGF and MMP-2 expression in skeletal muscle endothelial cells.

<p>Skeletal muscle endothelial cells were stimulated for 2 hours with Ang II (0.1 µM) and lysed for qPCR analysis of VEGF (A) and MMP-2 (C) transcript levels (n = 6 per condition). In (B), endothelial cells were treated overnight with Ang II (0.1 µM) and Western blotting was performed to assess VEGF protein levels, which were normalized to tubulin (n = 3 per condition). Endothelial cells were stimulated for either 10 or 30 minutes with 1 µM Ang II and then lysed for protein analysis. P-ERK1/2 (D) and P-Akt (E) levels were assessed by Western blotting and normalized to ß-actin and total Akt levels respectively (n = 3 per condition). Values are presented as mean ± SEM. One way ANOVA followed by Tukey’s multiple comparison test and student’s t-test were used to assess statistical significance (p<0.05). In panels A–D, *denotes p<0.05 vs. untreated cells. C – Control, 10–10 minutes and 30–30 minutes.</p

Contribution of endogenous Ang II to muscle VEGF production and endothelial cell-muscle cell crosstalk.

<p>C2C12 myotubes and confluent cultures of microvascular endothelial cells were lysed, and endogenous basal levels of angiotensinogen, AT1R and AT2R were assessed by Western blot analysis (A). In all blots, two independent cultures are shown for each cell type. C2C12 myotubes (n = 3 per condition) were treated overnight with the AT1R inhibitor Losartan (0.1 µM or 1 µM) and VEGF transcript levels were assessed by qPCR (B). Representative image and the quantification of RT-PCR analysis of VEGFA isoform expression (VEGF₁₂₀ ∼170 bp; VEGF₁₆₄ ∼300 bp and VEGF₁₈₀ = not detected) in C2C12 myotubes and skeletal muscle endothelial cells (C). Two independent samples are shown for both C2C12 and endothelial cells (n = 6 for C2C12 and n = 13 for endothelial cells). Lysates of matrix bound proteins were assessed for VEGF and tubulin protein expression by Western blotting (D). Cell extract (Ex) was used as a comparator with extracts of matrix (M) alone. Three independent samples of matrix-associated protein extracts are shown. Tubulin was detectable within cell extract, but not in matrix-derived extracts. In (E), endothelial cells alone or previously treated with an inhibitor of the VEGFR2, were incubated on matrix which previously contained C2C12 myoblasts with or without losartan treatment. Representative image and quantification of phosphorylated p38 levels (n = 6 for E and E+M, n = 5 for E+M_L and n = 4 for E_V+M). Values are presented as mean ± SEM. One way ANOVA followed by Tukey’s multiple comparison test and student’s t-test were used to assess statistical significance which was set as p<0.05. In panel B, *denotes p<0.05 vs. untreated cells. In panel C, *denotes p<0.05 vs. C2C12 cells and in panel E, *denotes p<0.05 vs. endothelial cells. E – endothelial cell, Ex - cell extract, Coll- type I collagen matrix, M - matrix lysate, E+M – endothelial cells + matrix from untreated C2C12, E+M_L – endothelial cells + matrix from losartan-treated C2C12 cells and E_V+M – endothelial cells pretreated with a VEGFR2 inhibitor + matrix from untreated C2C12.</p

Effect of skeletal muscle overload on angiotensinogen and AT receptor expression in skeletal muscle.

<p>Muscle subjected to overload for 5 days was lysed for qPCR analysis of angiotensinogen (A) and AT1aR (B) transcript levels. Protein levels of Angiotensinogen (C), AT1R (D) and AT2R (E) were assessed by Western Blotting. In each graph, values are presented as mean ± SEM (n = 3 for Sham and n = 6 for Overload). Student’s t-test (*) revealed a significant difference between angiotensinogen transcript and protein levels in overload compared to sham animals. OL – overload, Ang – angiotensinogen, AT1R – Ang II type 1 receptor and AT2R – Ang II type II receptor.</p