VPB1277 - Selective angiostatin neutralization promotes endothelial migration an early stage of angiogenesis

Tuesday, July 12, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: Virtual

Presenting Author(s)

Max S. Saito, PhD

Postdoctoral Researcher
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada
Edmonton, Alberta, Canada

Co-Author(s)

PJ

Paul Jurasz

Professor
Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada
Edmonton, Alberta, Canada

Background: Cardiovascular diseases are the primary cause of morbidity and mortality worldwide, and many coronary artery disease and peripheral artery disease patients are not eligible for standard therapies. Hence, therapeutic angiogenesis has been a sought-after experimental treatment strategy. Its limitation has been the sole focus on the delivery of pro-angiogenic factors and/or endothelial progenitor/stem cells, and not on concomitant suppression of counteracting endogenous negative angiogenesis regulators such as angiostatin. As such, our lab recently synthetized neutralizing peptides derived from ATP synthase, a known angiostatin target, to counteract the effects of angiostatin in an effort to promote therapeutic angiogenesis.

Aims: To investigate whether angiostatin neutralization promotes angiogenesis.

Methods: In vitro experiments were performed using Human Cardiac Microvascular Endothelial Cells (HMVEC-C). HMVEC-C were incubated with angiostatin (30 µg/mL) and neutralizing peptides (Alfa1-3 or non-neutralizing delta; 3uM) under hypoxia conditions (95% N2/5% CO2) for 48 hours. HMVEC-C endothelial nitric oxide synthase (eNOS), matrix metalloproteinase-14 (MMP-14) and MMP-2 levels were measured using immunoblot, and zymography as molecular markers of angiogenesis. HMVEC-C nitric oxide (NO) production was measured using DAF-FM-based flow cytometry and migration was assayed using a MMP-dependent modified Boyden chamber model.

Results: Angiostatin decreased HMVEC-C eNOS, MT1-MMP, and MMP-2 proteins levels, while only the combined incubation of alfa peptides 1-3 reversed angiostatin’s effects (eNOS: 91.5 ± 7.5%; MMP-14 104.5 ± 5.3%; MMP-2: 83.7± 17.1% of control, respectively). Similarly, angiostatin decreased HMVEC-C NO production and HMVEC-C migration, and these effects of angiostatin were reversed by the combined effects of alfa peptides 1-3 (NO production: 93.5 ± 15.8% and migration 93.3 ± 17.5% of control, respectively).

Conclusion(s): Combined the Alfa 1-3 peptides counteracted the effects of angiostatin, increasing eNOS, MMP-14 and, MMP2 levels, and NO production leading to increased HMVEC-C migration; thus, demonstrating their potential for their further study in in vivo ischemic models.