Vascular endothelial growth factor delays onset of failure in pressure-overload hypertrophy through matrix metalloproteinase activation and angiogenesis - PubMed (original) (raw)

Vascular endothelial growth factor delays onset of failure in pressure-overload hypertrophy through matrix metalloproteinase activation and angiogenesis

I Friehs et al. Basic Res Cardiol. 2006 May.

Abstract

Objective: Pressure-overload hypertrophy is associated with decreased capillary density in myocardium resulting in impaired substrate delivery. Treatment of hypertrophied hearts with vascular endothelial growth factor (VEGF) induces angiogenesis. Since angiogenesis is associated with extracellular matrix degradation, we sought to determine whether VEGF induced angiogenesis in hypertrophy required matrix metalloproteinases (MMP) activation.

Methods: Newborn rabbits underwent aortic banding. Progression of hypertrophy (mass-to-volume (M/V) ratio) and mid-wall contractility index was monitored by echocardiography. At 4 and 6 weeks, VEGF (2 microg/kg), vehicle or VEGF combined with GM6001 (5 mg/kg), a MMP inhibitor, was administered intrapericardially. CD-31 (indicator of angiogenesis), MMP-2, MT1-MMP and TIMPs (endogenous MMP inhibitors) expression were measured by immunoblotting. MMP-2 activity was determined by gelatin zymography.

Results: Untreated hypertrophied hearts progressed to ventricular dilatation at 7 wks (M/V ratio: 0.75 +/- 0.07), but compensatory hypertrophy was maintained with VEGF (0.91 +/- 0.07; p < 0.05). LV contractility declined in untreated hearts from -0.41 +/- 0.9 (5 wks) to -0.73 +/- 0.5 (7 wks; p < 0.05) but remained normal with VEGF (+1.61 +/- 0.6 vs. +0.47 +/- 0.2). MMP-2 expression and activity were significantly elevated in VEGF treated hypertrophied hearts (p < 0.05) and were blocked by concomitant administration of GM6001. VEGF induced neovascularization was inhibited by addition of GM6001. MT1-MMP showed a trend to higher levels in VEGF treated hearts. TIMPs were unchanged in all three groups.

Conclusions: Exogenous VEGF and resultant MMP-2 activation leads to increased capillary formation in severe hypertrophy, preventing progression to ventricular dilation and dysfunction. VEGF and the associated MMP-2 activation play an important and potentially therapeutic role in vascular remodeling of hypertrophied hearts.

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Figures

Fig. 1

Fig. 1

Microvascular density. A Representative immunoblots for CD-31 and summary of the densitometry data are depicted. VEGF treatment of hypertrophied hearts results in significant higher CD-31 levels. Concomitant administration of VEGF with a MMP inhibitor inhibited neoangiogenesis; however, the difference did not reach a level of significance (*p < 0.05 vs. VEGF treated hypertrophy). B Representative immunohistochemical sections of LV tissue are depicted with staining of the microvasculature with CD-31 in red or lectin in green

Fig. 2

Fig. 2

Left ventricular mass to volume ratio and midwall contractility. A LV mass to LV cavity volume measurement as an indicator of hypertrophic growth. VEGF treated hearts maintained a higher ratio of LV mass to cavity volume over an extended period compared to untreated hypertrophied hearts, which showed signs of severe ventricular dilatation (= fall in M/V ratio) (*p < 0.05; versus untreated hypertrophied hearts). B Midwall contractility (depicted as Z-scores) was calculated based on echocardiographic measurements of non-hypertrophied control hearts. VEGF treatment prevented myocardial dysfunction seen in the untreated hypertrophied hearts (*p < 0.05; versus untreated hypertrophied hearts)

Fig. 3

Fig. 3

MMP-2 activity. A, B MMP-2 activity levels were determined by gelatin zymography. Two representative gels are depicted with MMP activity for hypertrophied hearts and VEGF treated hearts at two different time points following treatment. Active MMP-2 was higher in VEGF treated hypertrophied hearts, one week following VEGF administration compared to untreated hypertrophied hearts. C A representative zymogram is shown which indicates that concomitant administration of VEGF and a MMP inhibitor decreased zymographic activity of MMP-2

Fig. 4

Fig. 4

Myocardial MMP protein content. A To reconfirm that MMP-2 is activated within one week following VEGF treatment, we performed immunoblot analysis using an antibody identifying total and active MMP-2. A representative immunoblot for this specific MMP species is shown for immunoprecipitates obtained from left ventricular muscle extracts from controls, untreated hypertrophied hearts and VEGF treated hypertrophied hearts. A distinct immunoreactive band could be localized at 72 kDa indicative of the latent, non-active form and a second one at 66 kDa which represents the active form of MMP-2. B, C Quantification of protein content of latent (B) and active (C) form was performed by laser densitometry and values are expressed as arbitrary densitometry units. There is no difference of latent MMP-2 protein content between controls (solid bar) and VEGF treated hypertrophied hearts (shaded bar) and untreated hypertrophied hearts (blank bar) but active MMP-2 levels were significantly higher in hypertrophied hearts following VEGF treatment (*p < 0.05; versus untreated hypertrophy and Control)

Fig. 5

Fig. 5

MT1-MMP protein content. AA representative immunoblot of MT1-MMP protein from left ventricular muscle extracts for all three groups is shown. BA summary of densitometry data showed that VEGF treatment resulted in an increase of MT1-MMP protein levels in hypertrophied hearts compared to untreated hearts but did not reach significance (p = 0.07)

Fig. 6

Fig. 6

TIMP levels in myocardium. A–D Representative immunoblots for all four TIMPs and summary of the densitometry data are depicted. There was no significant difference in TIMP protein levels between the groups

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