MMP-2/TIMP-2/TIMP-4 versus MMP-9/TIMP-3 in transition from compensatory hypertrophy and angiogenesis to decompensatory heart failure

Abstract

Although matrix metalloproteinase (MMPs) and tissue inhibitor of metalloproteinase (TIMPs) play a vital role in tumour angiogenesis and TIMP-3 caused apoptosis, their role in cardiac angiogenesis is unknown. Interestingly, a disruption of co-ordinated cardiac hypertrophy and angiogenesis contributed to the transition to heart failure, however, the proteolytic and anti-angiogenic mechanisms of transition from compensatory hypertrophy to decompensatory heart failure were unclear. We hypothesized that after an aortic stenosis MMP-2 released angiogenic factors during compensatory hypertrophy and MMP-9/TIMP-3 released anti-angiogenic factors causing decompensatory heart failure. To verify this hypothesis, wild type (WT) mice were studied 3 and 8 weeks after aortic stenosis, created by banding the ascending aorta in WT and MMP-9-/- (MMP-9KO) mice. Cardiac function (echo, PV loops) was decreased at 8 weeks after stenosis. The levels of MMP-2 (western blot) increased at 3 weeks and returned to control level at 8 weeks, MMP-9 increased only at 8 weeks. TIMP-2 and -4 decreased at 3 and even more at 8 weeks. The angiogenic VEGF increased at 3 weeks and decreased at 8 weeks, the anti-angiogenic endostatin and angiostatin increased only at 8 weeks. CD-31 positive endothelial cells were more intensely labelled at 3 weeks than in sham operated or in 8 weeks banded mice. Vascularization, as estimated by x-ray angiography, was increased at 3 weeks and decreased at 8 weeks post-banding. Although the vast majority of studies were performed on control WT mice only, interestingly, MMP9-KO mice seemed to have increased vascular density 8 weeks after banding. These results suggested that there was increase in MMP-2, decrease in TIMP-2 and -4, increase in angiogenic factors and vascularization in compensatory hearts. However, in decompensatory hearts there was increase in MMP-9, TIMP-3, endostatin, angiostatin and vascular rarefaction.

Figure 1

Panels represented M-mode echocardiography of Sham (8wks), 3 wks and 8 wks after aortic banding. The bars graphs represented % fraction shortening (FS), LVIDd (left ventricular internal dimension in diastole), and LVPWd (left ventricular posterior wall dimension in diastole). *, p<0.05 compared with sham. All bar graphs depicted mean+SE from n=5/group

Figure 2

Pressure-volume loop by Millar catheter of Sham (8wks) and 8 wks aortic banding mice: The bar graphs depict % ejection fraction (EF), ESV (End systolic stroke volume), and stroke volume (SV) in sham and 8 wks aortic banding mice. The data presented mean±SE from n=5/group *, p<0.05 vs sham. PV analysis in vivo is load-dependent, that changes in after load and preload after banding can influence the parameters independently of heart failure at 8 wks.

Figure 3

The gross images of the whole heart of Sham (8wks), 3 wks, and 8 wks aortic banding. Hearts were cleansed in phosphate buffered saline. The left ventricle was separated. The ratio of left ventricle (LV) weight (wt) to body weight (BW) was depicted in bar graph. The data presented mean±SE, n=5/group. *, p<0.01 vs Sham.

Figure 4

Representative microphotographs of mason’s-trichrome blue histological staining of frozen heart sections: The hearts from sham (8wks), 3 wks and 8 wks aortic banding were analyzed. Arrow indicates endocardial and pericapillary fibrosis. 20 x magnifications. The insert box, 100 x magnifications.

Figure 5

Western blot analyses of MMP-9 and MMP-2 expression in Sham (8wks), 3 wks and 8 wks of aortic banding: Bar graphs showed densitometric analyses of MMP-9 and MMP-2 expression over sham groups (protein normalized with GAPDH). Each bar represents mean ± SE, *p<0.05 vs sham, **, compared with 3 wks, from n=5 in each group. The % protein expression on Y-axis is the protein expression calculated as fold change vs. sham.

Figure 6

Western blot analyses of TIMP-2 and TIMP-4 expression in Sham (8wks), 3 wks and 8 wks of aortic banding: Bar graphs showed densitometric analyses of TIMP-2 and TIMP-4 expression over sham groups (protein normalized with GAPDH). Each bar represents mean ± SE, *p<0.05 vs sham, **, compared with 3 wks, from n=5 in each group. The % protein expression on Y-axis is the protein expression calculated as fold change vs. sham.

Figure 7

Left ventricle immunohistochemical staining and co-localization of TIMP-3 and MMP-2: Cryocut frozen sections of (8–10 μm) were stained and secondarily conjugated with FITC for TIMP-3 and texas red for MMP-2 (white arrows indicate capillaries). The bar diagrams depicted the intensity quantification of TIMP-3 and MMP-2 by Fluoview software, in sham (8wks), 3 wks and 8 wks aortic banding. Data presented mean ± SE; *p<0.01 vs sham; **p<0.05 vs 3 wks; n=5 animals per group.

Figure 8

Measurement of angiogenic and anti-angiogenic factors: Western blot analyses of VEGF-A, endostatin and angiostatin. Bar graphs showed the relative expression of VEGF-A, endostatin, and angiostatin over sham group (after normalization with GAPDH). The data presented mean±SE, * p<0.05 vs sham; **, compared with 3wks. The % protein expression on Y-axis is the protein expression calculated as fold change vs. sham.

Figure 9

Immunohistochemical staining of endostatin. Cryocut frozen tissue sections (5 μm) are stained with anti-endostation antibody secondarily conjugated with Texas red dye. Nuclei are stained with DAPI. Bar diagrams depicted intensity quantification by image pro-software. Each bar representative mean±SE, **p<0.05 vs sham or 3 wks, n=5 animals/group.

Figure 10

Immunohistochemical staining with CD31 (PECAM) in sham (8wks), 3 wks and 8 wks aortic banding groups. DAPI was used for nuclear staining. The images were merged. The arrows point to capillary structure. Bar diagrams represent quantified arbitrary number of CD 31staining endothelial cell in capillaries per each field randomly selected with the help of Image-Pro software. The data presented mean±SE, n=5/group. * p<0.01 vs Sham; **p<0.05 vs 3 wks.

Figure 11

Barium-contrast x-ray angiograms, to demonstrate gross variability in vasculature among sham (8wks), 3 wks, 8 wks and MMP-9KO (at 8 wks). In vivo hearts were perfused with Barium sulfate solution and x-ray imaging was taken with KODAK MM4000 in both in whole body and in isolated hearts. The bar diagrams depicted quantified intensity of the images from randomly chosen areas of same size, with Image-J software. The data presented mean±SE, *p<0.05 vs sham; **p<0.01 vs 3 wks; ***, compared with 8wks. n=5 animals per group.

Figure 12

Hypothetical presentation of latent MMP activation under chronic oxidative load condition. The imbalance in MMP/TIMP axis and remodeling, generating angiogenic, anti-angiogenic, factors, leading to apoptosis and chronic heart failure (CHF). Abbreviations: ROS, reactive oxygen species; RNS, reactive nitrogen species; RTS, reactive thiol species; NADH, nicotinamide adenine dinucleotide (reduced); SOD, superoxide dismutase; Prx, peroxiredoxin; VEGF, vascular endothelial growth factor; ADAM, a disintegrin and metalloproteinase; Meprins, metalloproteinase in renal; iNOS, inducible nitric oxide synthase; eNOS, endothelial nitric oxide synthase; GSSG/GSH, oxidized glutathione/reduced glutathione; mtMMP, mitochondrial MMP; MT-MMP, membrane type-MMP.