A Relationship Between Vascular Endothelial Growth Factor, Angiogenesis, and Cardiac Repair After Muscle Stem Cell Transplantation Into Ischemic Hearts
Thomas R. Payne, PhD*, , ,
Hideki Oshima, MD, PhD*,,
Masaho Okada, MD*,,
Nobuo Momoi, MD ,
Kimimasa Tobita, MD,
Bradley B. Keller, MD,
Hairong Peng, MD, PhD*, and
Johnny Huard, PhD*,,,||,*
* Stem Cell Research Center, Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania
Pediatric Cardiovascular Research Program, Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania
Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania.
Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania.
|| Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, Pennsylvania.
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Figure 1 VEGF-Induced Infarct Neovascularization
Hearts in the muscle-derived stem cell (MDSC)-LacZ, MDSC-vascular endothelial growth factor (VEGF)25, and MDSC-VEGF50 groups contained the most blood vessels per high-power field (HPF) (200x magnification) in the infarct area, and significantly more than the phosphate-buffered saline (PBS)- or MDSC-FLT–injected hearts (*p < 0.01 vs. PBS; p < 0.01 vs. MDSC-FLT). Hearts in the MDSC-FLT group displayed comparable vascularity to the control PBS-injected hearts. The MDSC-VEGF25 and MDSC-VEGF50 groups displayed higher capillary densities than the MDSC-LacZ group (p < 0.01 vs. MDSC-LacZ). The MDSC-VEGF25 group displayed comparable vascularity to the MDSC-VEGF50 group.
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Figure 2 Abnormal Angiogenesis
Overexpression of VEGF in the MDSC-VEGF50–injected hearts formed disorganized vasculature (CD31 immunostaining, white) 12 weeks after injection. In contrast, we observed normal spatially organized vasculature in hearts transplanted with MDSCs expressing lower levels of the VEGF transgene (MDSC-VEGF25 group) or control MDSCs (MDSC-LacZ) (200x magnification). Abbreviations as in Figure 1.
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Figure 3 Effect of VEGF on Infarct Scar Size
(A and B) Hearts injected with MDSC-LacZ and MDSC-VEGF25 cells displayed smaller infarct sizes and scar tissue ratio compared with hearts injected with PBS or MDSC-FLT cells at all time points (*p < 0.05 vs. PBS; p < 0.05 vs. MDSC-FLT). Although hearts injected with MDSC-VEGF50 cells had smaller scars at 2 and 6 weeks, they demonstrated larger infarct sizes and scar tissue ratio at 12 weeks (p < 0.05 vs. MDSC-VEGF50 12 weeks; #p < 0.05, 2 vs. 12 weeks). LV = left ventricle; other abbreviations as in Figure 1.
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Figure 4 Antagonism of VEGF Diminished the Therapeutic Effect of MDSC Transplantation
(A) Representative echocardiographic M-mode images of left ventricle for each group. End-systolic dimension (ESD) and end-diastolic dimension (EDD) were measured in each image. (B) Hearts injected with PBS, MDSC-FLT cells, or MDSC-VEGF50 cells displayed progressive enlargement of the left ventricle, as measured by EDD, over the course of the study (#p < 0.05, 2 vs. 12 weeks), and these left ventricle dimensions were larger than the MDSC-LacZ- and MDSC-VEGF25–injected hearts at all time points (p < 0.05 vs. MDSC-LacZ and MDSC-VEGF25). In contrast, left ventricle dimensions in the MDSC-LacZ– and MDSC-VEGF25–injected hearts were preserved. (C and D) Injection of MDSC-LacZ, MDSC-VEGF25, or MDSC-VEGF50 cells improved left ventricle contractility (% fractional shortening, % fractional area change) compared with the injection of PBS and MDSC-FLT cells (*p < 0.05 vs. PBS; p < 0.05 vs. MDSC-FLT). Systolic function in the PBS, MDSC-FLT, and MDSC-VEGF50 groups decreased by 12 weeks after injection (#p < 0.05, 2 vs. 12 weeks; p < 0.05 MDSC-VEGF50 vs. MDSC-LacZ and MDSC-VEGF25 groups), whereas systolic function in the MDSC-LacZ– and MDSC-VEGF25–injected hearts was preserved. Abbreviations as in Figure 1.
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Figure 5 MDSCs Secrete VEGF Under Hypoxia and Mechanical Stimulation
(A) Compared with MDSCs cultured under normoxia (in proliferation medium [PM]), MDSCs exposed to hypoxic conditions (2.5% oxygen) exhibited a 6-fold increase in VEGF secretion (in PM), and a 9-fold increase if grown in serum-free (SF) medium (*p < 0.05 vs. normoxia and PM or SF). Muscle-derived stem cells cultured under hypoxic conditions in SF-medium secreted the most VEGF. (B) When subjected to cyclic stretch for 10 and 24 h, MDSCs increased VEGF expression 2-fold (*p < 0.05 vs. non-stretch control group). Abbreviations as in Figure 1.
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