New categorization of human vascular endothelial cells by pro- vs anti-proliferative phenotypes

Abstract

AIM: To integrally understand the effects of human vascular endothelial cells (VECs) on the proliferation of vascular smooth muscle cells (VSMCs).

METHODS: Various kinds of human VECs of different origins were co-cultured with human aortic smooth muscle cells, a representative of human VSMCs. To exclude the irrelevant effects due to growth competition between VECs and VSMCs, the proliferation of VECs had previously been arrested via a low-dose gamma ray irradiation. To discriminately analyze the proliferation of VSMCs from that of VECs, the former cells were labeled with red fluorescent dye while the latter cells were labeled with green fluorescent dye before performing co-culture experiments. After 4 d, total cells were harvested and subjected to flow cytometric analyses. Decrements in red fluorescence intensities due to proliferation-mediated dilutions were measured and mathematically processed using a specific software to quantitatively evaluate the proliferation of VSMCs. The findings obtained from the flow cytometry-based analyses were further validated by microscopic observations.

RESULTS: Commercially available primary cultured human VECs exclusively promoted VSMC proliferation regardless of their tissue origins and we termed these pro-proliferative VECs as “type-I”. By contrast, VECs freshly generated from human bone marrow-derived endothelial progenitors cells or human pluripotent stem cells including embryonic stem cells and induced pluripotent stem cells suppressed VSMC proliferation and we termed these anti-proliferative VECs as “type-II”. Repetitive subcultures as well as oxidative stress induced “type-II VECs to type-I” conversion along with an induction of Regulator of G-protein signaling 5 (RGS5). Compatibly, anti-oxidant treatments suppressed both the subculture-dependent “type-II to type-I” conversion and an induction of RGS5 gene. Immunostaining studies of clinical specimens indicated that RGS5 protein expressions in endothelial layers were low in normal arteries but they were up-regulated in pathological arteries including hypertension, atherosclerosis and autoimmune vasculitis in a dose-dependent manner. Overexpression and knockdown of RGS5 caused that “type-II to type-I” and “type-I to type-II” phenotype conversions of VECs, respectively.

CONCLUSION: Human VECs are categorized into two types: pro-proliferative RGS5^high VECs (type-I) and anti-proliferative RGS5^low VECs (type-II).

Keywords: Vascular endothelial cells, Vascular smooth muscle cells, Human induced pluripotent stem cells, Human embryonic stem cells, Regulator of G-protein signaling 5, Oxidative stress

Core tip: There is a longstanding controversy over the effects of vascular endothelial cells (VECs) on the proliferation of vascular smooth muscle cells (VSMCs). Since the controversy came from lack of systematic studies, we performed an integrated analysis using various human VECs to quantitatively evaluate their effects on VSMC proliferation. Here we report that: (1) human VECs are classified into two groups: pro-proliferative (type-I) vs“anti-proliferative” (type-II); (2) oxidative stress and ageing induced “type-II to type-I” conversion; and (3) RGS5 is the responsible gene for VEC phenotype determinations. Thus, human VECs are categorized into pro-proliferative RGS5^high (type-I) and anti-proliferative RGS5^low (type-II) VECs.