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Moderation of calpain activity promotes neovascular integration and lumen formation during VEGF-induced pathological angiogenesis.

Hoang MV, Nagy JA, Fox JE, Senger DR - PLoS ONE (2010)

Bottom Line: Moderate doses of calpain inhibitor-I improved VEGF-driven angiogenesis similarly to DN calpain-I.Consistent with the critical importance of microtubules for vascular network integration, the microtubule-stabilizing agent taxol supported vascular cord integration whereas microtubule dissolution with nocodazole collapsed cord networks.These findings implicate VEGF-induction of calpain activity and impairment of cytoskeletal dynamics in the failure of VEGF-induced neovessels to form and integrate properly.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Center for Vascular Biology Research, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: Successful neovascularization requires that sprouting endothelial cells (ECs) integrate to form new vascular networks. However, architecturally defective, poorly integrated vessels with blind ends are typical of pathological angiogenesis induced by vascular endothelial growth factor-A (VEGF), thereby limiting the utility of VEGF for therapeutic angiogenesis and aggravating ischemia-related pathologies. Here we investigated the possibility that over-exuberant calpain activity is responsible for aberrant VEGF neovessel architecture and integration. Calpains are a family of intracellular calcium-dependent, non-lysosomal cysteine proteases that regulate cellular functions through proteolysis of numerous substrates.

Methodology/principal findings: In a mouse skin model of VEGF-driven angiogenesis, retroviral transduction with dominant-negative (DN) calpain-I promoted neovessel integration and lumen formation, reduced blind ends, and improved vascular perfusion. Moderate doses of calpain inhibitor-I improved VEGF-driven angiogenesis similarly to DN calpain-I. Conversely, retroviral transduction with wild-type (WT) calpain-I abolished neovessel integration and lumen formation. In vitro, moderate suppression of calpain activity with DN calpain-I or calpain inhibitor-I increased the microtubule-stabilizing protein tau in endothelial cells (ECs), increased the average length of microtubules, increased actin cable length, and increased the interconnectivity of vascular cords. Conversely, WT calpain-I diminished tau, collapsed microtubules, disrupted actin cables, and inhibited integration of cord networks. Consistent with the critical importance of microtubules for vascular network integration, the microtubule-stabilizing agent taxol supported vascular cord integration whereas microtubule dissolution with nocodazole collapsed cord networks.

Conclusions/significance: These findings implicate VEGF-induction of calpain activity and impairment of cytoskeletal dynamics in the failure of VEGF-induced neovessels to form and integrate properly. Accordingly, calpain represents an important target for rectifying key vascular defects associated with pathological angiogenesis and for improving therapeutic angiogenesis with VEGF.

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VEGF increases calpain activity in MVECs, and calpain inhibitor-I improves VEGF angiogenesis.(A) MVECs, cultured in the absence of VEGF, were incubated with fluorescent calpain substrate (see Methods) and stimulated with 20 ng/ml VEGF for 15 min; n≥10; control vs. VEGF (p<0.001), VEGF vs. VEGF plus 200 nM calpastatin peptide (p<0.002), VEGF vs. VEGF plus 200 nM ALLN (p<0.002). (B) VEGF-stimulated MVECs undergoing capillary morphogenesis in 3D collagen. Reduction of calpain activity to normal baseline levels with 200 nM calpain inhibitor-I reduced blind ends (white arrows) and markedly improved integration of cord networks. Bar  = 50 µm. (C) Quantification of cord assays shown in (B); n≥15. Measured parameters correspond to values for samples areas of 0.4 mm2. Calpain inhibitor-I at the 200 nM dose had no effect on EC density but strongly improved vascular network integration, as indicated by >100% increase in average cord length (p<0.003), >50% reduction in blind ends (p<0.001), and nearly 50% increase in polygons, i.e. closed networks (p<0.005). (D) Daily systemic administration of calpain inhibitor-I (10 mg/kg) improves integration and perfusion of new blood vessels. Skin angiogenesis was provoked by VEGF as in Figure 1 but without retroviral packaging cells. Instead animals were treated daily, beginning on day 2, with 10 mg/kg calpain inhibitor-I and harvested on day 8. Evans Blue Dye: images of dermis overlying the Matrigel implants (scale bar  = 250 microns) following perfusion with dye for 10 min, illustrating that calpain inhibitor-I improved blood vessel integration and perfusion (blue color) and reduced blind ends (arrows) relative to vehicle control. CD31 Staining: ECs in cross section stained with CD31 antibody (brown color) illustrating that calpain inhibitor-I improves lumen formation (arrows) relative to control. Scale bar  = 30 microns. S  =  smooth muscle, V  =  region of neovascularization, M  =  Matrigel. (E) Quantification of new blood vessel density, closed vascular networks (polygons), and blind ends from gross images, and EC density and relative lumen area in cross-section from paraffin sections stained with CD31 antibody; n≥17. Gross vessel density (p<0.01), polygons (p<0.03), blind ends (p<0.01), relative lumen area (p<0.01). Numbers of polygons and blind ends correspond to sample areas of 10 mm2.
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pone-0013612-g004: VEGF increases calpain activity in MVECs, and calpain inhibitor-I improves VEGF angiogenesis.(A) MVECs, cultured in the absence of VEGF, were incubated with fluorescent calpain substrate (see Methods) and stimulated with 20 ng/ml VEGF for 15 min; n≥10; control vs. VEGF (p<0.001), VEGF vs. VEGF plus 200 nM calpastatin peptide (p<0.002), VEGF vs. VEGF plus 200 nM ALLN (p<0.002). (B) VEGF-stimulated MVECs undergoing capillary morphogenesis in 3D collagen. Reduction of calpain activity to normal baseline levels with 200 nM calpain inhibitor-I reduced blind ends (white arrows) and markedly improved integration of cord networks. Bar  = 50 µm. (C) Quantification of cord assays shown in (B); n≥15. Measured parameters correspond to values for samples areas of 0.4 mm2. Calpain inhibitor-I at the 200 nM dose had no effect on EC density but strongly improved vascular network integration, as indicated by >100% increase in average cord length (p<0.003), >50% reduction in blind ends (p<0.001), and nearly 50% increase in polygons, i.e. closed networks (p<0.005). (D) Daily systemic administration of calpain inhibitor-I (10 mg/kg) improves integration and perfusion of new blood vessels. Skin angiogenesis was provoked by VEGF as in Figure 1 but without retroviral packaging cells. Instead animals were treated daily, beginning on day 2, with 10 mg/kg calpain inhibitor-I and harvested on day 8. Evans Blue Dye: images of dermis overlying the Matrigel implants (scale bar  = 250 microns) following perfusion with dye for 10 min, illustrating that calpain inhibitor-I improved blood vessel integration and perfusion (blue color) and reduced blind ends (arrows) relative to vehicle control. CD31 Staining: ECs in cross section stained with CD31 antibody (brown color) illustrating that calpain inhibitor-I improves lumen formation (arrows) relative to control. Scale bar  = 30 microns. S  =  smooth muscle, V  =  region of neovascularization, M  =  Matrigel. (E) Quantification of new blood vessel density, closed vascular networks (polygons), and blind ends from gross images, and EC density and relative lumen area in cross-section from paraffin sections stained with CD31 antibody; n≥17. Gross vessel density (p<0.01), polygons (p<0.03), blind ends (p<0.01), relative lumen area (p<0.01). Numbers of polygons and blind ends correspond to sample areas of 10 mm2.

Mentions: Consistent with previous reports that VEGF induces calpain activity in MVECs from lung and skin [13], [14], we found that VEGF, at the concentration routinely employed in all of our in vitro experiments (20 ng/ml), induced calpain activity >50% (Fig. 4A). Calpastatin peptide (200 nM) and another calpain inhibitor, calpain inhibitor-I, also known as ALLN (200 nM), each reduced calpain activity in VEGF-stimulated cells to levels present in the absence of VEGF stimulation. Moreover, 200 nM ALLN, which suppressed calpain activity to baseline identically with 200 nM calpastatin peptide (Fig. 4A), markedly improved integration of vascular cords as measured by reduction in blind ends and increased network connectivity (Fig. 4B, C). However, higher doses of ALLN (≥2.0 µM) did not improve integration of cords, but rather these doses were inhibitory and caused cell rounding (not shown). Notably, these higher doses of ALLN (≥2 µM) severely inhibited calpain activity in comparison with 200 nM ALLN (Fig. 4A), underscoring the importance of moderate calpain inhibition to achieve the desired outcome. To summarize, these experiments indicate that: (1) VEGF induction of calpain activity is likely responsible for impaired integration of vascular cords, and (2) that normalization of calpain activity to baseline levels with calpain inhibitor-I, improves integration of vascular cords comparably to DN calpain-I (Fig. 2). Moreover, these findings suggested that systemic administration of this inhibitor might similarly improve neovascularization in vivo. To test this possibility, we employed the same VEGF-driven angiogenesis model described above but without retroviral packaging cells. Instead animals were treated with calpain inhibitor-I (ALLN), which has been used extensively in animal models but for other applications [28], [29], [30], [31]. In initial pilot experiments, calpain inhibitor-I was administered daily (5, 10, 15, and 20 mg/kg, i.p.) beginning on day two following implantation of the VEGF-transfectants. No adverse effects on animal health were observed with any of these doses. As determined grossly in these pilot experiments at day 8, the 20 mg/kg dose clearly inhibited angiogenesis. In contrast, the 10 mg/kg dose did not inhibit neovascularization but rather improved the integration of neovessels. Therefore, more extensive experiments and analyses were performed with the 10 mg/kg dose. As quantified grossly and in cross-section (Figure 4 D, E), 10 mg/kg daily calpain inhibitor-I markedly reduced blind ends and increased vessel lumens similarly to DN calpain-I. These improvements in neovascular architecture were accomplished without any detectable effect on EC density (Figure 4E), indicating that daily administration of 10 mg/kg calpain inhibitor-I did not affect EC number. Similar to DN calpain-I, calpain inhibitor-I (200 nM, ∼1 x IC50) had no effect on production of VEGF by SK-MEL2 cells (the source of VEGF expression) (see Methods), consistent with the fact that VEGF expression was driven constitutively by a CMV promoter. Thus, the cumulative evidence indicates that improvement in neovascular architecture by calpain inhibitor-I is best explained by improvement in capillary morphogenesis rather than by differences in EC density or VEGF expression.


Moderation of calpain activity promotes neovascular integration and lumen formation during VEGF-induced pathological angiogenesis.

Hoang MV, Nagy JA, Fox JE, Senger DR - PLoS ONE (2010)

VEGF increases calpain activity in MVECs, and calpain inhibitor-I improves VEGF angiogenesis.(A) MVECs, cultured in the absence of VEGF, were incubated with fluorescent calpain substrate (see Methods) and stimulated with 20 ng/ml VEGF for 15 min; n≥10; control vs. VEGF (p<0.001), VEGF vs. VEGF plus 200 nM calpastatin peptide (p<0.002), VEGF vs. VEGF plus 200 nM ALLN (p<0.002). (B) VEGF-stimulated MVECs undergoing capillary morphogenesis in 3D collagen. Reduction of calpain activity to normal baseline levels with 200 nM calpain inhibitor-I reduced blind ends (white arrows) and markedly improved integration of cord networks. Bar  = 50 µm. (C) Quantification of cord assays shown in (B); n≥15. Measured parameters correspond to values for samples areas of 0.4 mm2. Calpain inhibitor-I at the 200 nM dose had no effect on EC density but strongly improved vascular network integration, as indicated by >100% increase in average cord length (p<0.003), >50% reduction in blind ends (p<0.001), and nearly 50% increase in polygons, i.e. closed networks (p<0.005). (D) Daily systemic administration of calpain inhibitor-I (10 mg/kg) improves integration and perfusion of new blood vessels. Skin angiogenesis was provoked by VEGF as in Figure 1 but without retroviral packaging cells. Instead animals were treated daily, beginning on day 2, with 10 mg/kg calpain inhibitor-I and harvested on day 8. Evans Blue Dye: images of dermis overlying the Matrigel implants (scale bar  = 250 microns) following perfusion with dye for 10 min, illustrating that calpain inhibitor-I improved blood vessel integration and perfusion (blue color) and reduced blind ends (arrows) relative to vehicle control. CD31 Staining: ECs in cross section stained with CD31 antibody (brown color) illustrating that calpain inhibitor-I improves lumen formation (arrows) relative to control. Scale bar  = 30 microns. S  =  smooth muscle, V  =  region of neovascularization, M  =  Matrigel. (E) Quantification of new blood vessel density, closed vascular networks (polygons), and blind ends from gross images, and EC density and relative lumen area in cross-section from paraffin sections stained with CD31 antibody; n≥17. Gross vessel density (p<0.01), polygons (p<0.03), blind ends (p<0.01), relative lumen area (p<0.01). Numbers of polygons and blind ends correspond to sample areas of 10 mm2.
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pone-0013612-g004: VEGF increases calpain activity in MVECs, and calpain inhibitor-I improves VEGF angiogenesis.(A) MVECs, cultured in the absence of VEGF, were incubated with fluorescent calpain substrate (see Methods) and stimulated with 20 ng/ml VEGF for 15 min; n≥10; control vs. VEGF (p<0.001), VEGF vs. VEGF plus 200 nM calpastatin peptide (p<0.002), VEGF vs. VEGF plus 200 nM ALLN (p<0.002). (B) VEGF-stimulated MVECs undergoing capillary morphogenesis in 3D collagen. Reduction of calpain activity to normal baseline levels with 200 nM calpain inhibitor-I reduced blind ends (white arrows) and markedly improved integration of cord networks. Bar  = 50 µm. (C) Quantification of cord assays shown in (B); n≥15. Measured parameters correspond to values for samples areas of 0.4 mm2. Calpain inhibitor-I at the 200 nM dose had no effect on EC density but strongly improved vascular network integration, as indicated by >100% increase in average cord length (p<0.003), >50% reduction in blind ends (p<0.001), and nearly 50% increase in polygons, i.e. closed networks (p<0.005). (D) Daily systemic administration of calpain inhibitor-I (10 mg/kg) improves integration and perfusion of new blood vessels. Skin angiogenesis was provoked by VEGF as in Figure 1 but without retroviral packaging cells. Instead animals were treated daily, beginning on day 2, with 10 mg/kg calpain inhibitor-I and harvested on day 8. Evans Blue Dye: images of dermis overlying the Matrigel implants (scale bar  = 250 microns) following perfusion with dye for 10 min, illustrating that calpain inhibitor-I improved blood vessel integration and perfusion (blue color) and reduced blind ends (arrows) relative to vehicle control. CD31 Staining: ECs in cross section stained with CD31 antibody (brown color) illustrating that calpain inhibitor-I improves lumen formation (arrows) relative to control. Scale bar  = 30 microns. S  =  smooth muscle, V  =  region of neovascularization, M  =  Matrigel. (E) Quantification of new blood vessel density, closed vascular networks (polygons), and blind ends from gross images, and EC density and relative lumen area in cross-section from paraffin sections stained with CD31 antibody; n≥17. Gross vessel density (p<0.01), polygons (p<0.03), blind ends (p<0.01), relative lumen area (p<0.01). Numbers of polygons and blind ends correspond to sample areas of 10 mm2.
Mentions: Consistent with previous reports that VEGF induces calpain activity in MVECs from lung and skin [13], [14], we found that VEGF, at the concentration routinely employed in all of our in vitro experiments (20 ng/ml), induced calpain activity >50% (Fig. 4A). Calpastatin peptide (200 nM) and another calpain inhibitor, calpain inhibitor-I, also known as ALLN (200 nM), each reduced calpain activity in VEGF-stimulated cells to levels present in the absence of VEGF stimulation. Moreover, 200 nM ALLN, which suppressed calpain activity to baseline identically with 200 nM calpastatin peptide (Fig. 4A), markedly improved integration of vascular cords as measured by reduction in blind ends and increased network connectivity (Fig. 4B, C). However, higher doses of ALLN (≥2.0 µM) did not improve integration of cords, but rather these doses were inhibitory and caused cell rounding (not shown). Notably, these higher doses of ALLN (≥2 µM) severely inhibited calpain activity in comparison with 200 nM ALLN (Fig. 4A), underscoring the importance of moderate calpain inhibition to achieve the desired outcome. To summarize, these experiments indicate that: (1) VEGF induction of calpain activity is likely responsible for impaired integration of vascular cords, and (2) that normalization of calpain activity to baseline levels with calpain inhibitor-I, improves integration of vascular cords comparably to DN calpain-I (Fig. 2). Moreover, these findings suggested that systemic administration of this inhibitor might similarly improve neovascularization in vivo. To test this possibility, we employed the same VEGF-driven angiogenesis model described above but without retroviral packaging cells. Instead animals were treated with calpain inhibitor-I (ALLN), which has been used extensively in animal models but for other applications [28], [29], [30], [31]. In initial pilot experiments, calpain inhibitor-I was administered daily (5, 10, 15, and 20 mg/kg, i.p.) beginning on day two following implantation of the VEGF-transfectants. No adverse effects on animal health were observed with any of these doses. As determined grossly in these pilot experiments at day 8, the 20 mg/kg dose clearly inhibited angiogenesis. In contrast, the 10 mg/kg dose did not inhibit neovascularization but rather improved the integration of neovessels. Therefore, more extensive experiments and analyses were performed with the 10 mg/kg dose. As quantified grossly and in cross-section (Figure 4 D, E), 10 mg/kg daily calpain inhibitor-I markedly reduced blind ends and increased vessel lumens similarly to DN calpain-I. These improvements in neovascular architecture were accomplished without any detectable effect on EC density (Figure 4E), indicating that daily administration of 10 mg/kg calpain inhibitor-I did not affect EC number. Similar to DN calpain-I, calpain inhibitor-I (200 nM, ∼1 x IC50) had no effect on production of VEGF by SK-MEL2 cells (the source of VEGF expression) (see Methods), consistent with the fact that VEGF expression was driven constitutively by a CMV promoter. Thus, the cumulative evidence indicates that improvement in neovascular architecture by calpain inhibitor-I is best explained by improvement in capillary morphogenesis rather than by differences in EC density or VEGF expression.

Bottom Line: Moderate doses of calpain inhibitor-I improved VEGF-driven angiogenesis similarly to DN calpain-I.Consistent with the critical importance of microtubules for vascular network integration, the microtubule-stabilizing agent taxol supported vascular cord integration whereas microtubule dissolution with nocodazole collapsed cord networks.These findings implicate VEGF-induction of calpain activity and impairment of cytoskeletal dynamics in the failure of VEGF-induced neovessels to form and integrate properly.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Center for Vascular Biology Research, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts, United States of America.

ABSTRACT

Background: Successful neovascularization requires that sprouting endothelial cells (ECs) integrate to form new vascular networks. However, architecturally defective, poorly integrated vessels with blind ends are typical of pathological angiogenesis induced by vascular endothelial growth factor-A (VEGF), thereby limiting the utility of VEGF for therapeutic angiogenesis and aggravating ischemia-related pathologies. Here we investigated the possibility that over-exuberant calpain activity is responsible for aberrant VEGF neovessel architecture and integration. Calpains are a family of intracellular calcium-dependent, non-lysosomal cysteine proteases that regulate cellular functions through proteolysis of numerous substrates.

Methodology/principal findings: In a mouse skin model of VEGF-driven angiogenesis, retroviral transduction with dominant-negative (DN) calpain-I promoted neovessel integration and lumen formation, reduced blind ends, and improved vascular perfusion. Moderate doses of calpain inhibitor-I improved VEGF-driven angiogenesis similarly to DN calpain-I. Conversely, retroviral transduction with wild-type (WT) calpain-I abolished neovessel integration and lumen formation. In vitro, moderate suppression of calpain activity with DN calpain-I or calpain inhibitor-I increased the microtubule-stabilizing protein tau in endothelial cells (ECs), increased the average length of microtubules, increased actin cable length, and increased the interconnectivity of vascular cords. Conversely, WT calpain-I diminished tau, collapsed microtubules, disrupted actin cables, and inhibited integration of cord networks. Consistent with the critical importance of microtubules for vascular network integration, the microtubule-stabilizing agent taxol supported vascular cord integration whereas microtubule dissolution with nocodazole collapsed cord networks.

Conclusions/significance: These findings implicate VEGF-induction of calpain activity and impairment of cytoskeletal dynamics in the failure of VEGF-induced neovessels to form and integrate properly. Accordingly, calpain represents an important target for rectifying key vascular defects associated with pathological angiogenesis and for improving therapeutic angiogenesis with VEGF.

Show MeSH
Related in: MedlinePlus