Cavin-2 regulates the activity and stability of endothelial nitric-oxide synthase (eNOS) in angiogenesis

Gandhi T.K Boopathy,Madhura Kulkarni,Sze Yuan Ho,Adrian Boey,Edmond Wei Min Chua,Veluchamy A Barathi,Tom J Carney,Xiaomeng Wang,Wanjin Hong

doi:10.1074/jbc.m117.794743

Abstract

Angiogenesis is a highly regulated process for formation of new blood vessels from pre-existing ones. Angiogenesis is dysregulated in various pathologies, including age-related macular degeneration, arthritis, and cancer. Inhibiting pathological angiogenesis therefore represents a promising therapeutic strategy for treating these disorders, highlighting the need to study angiogenesis in more detail. To this end, identifying the genes essential for blood vessel formation and elucidating their function are crucial for a complete understanding of angiogenesis. Here, focusing on potential candidate genes for angiogenesis, we performed a morpholino-based genetic screen in zebrafish and identified Cavin-2, a membrane-bound phosphatidylserine-binding protein and critical organizer of caveolae (small microdomains in the plasma membrane), as a regulator of angiogenesis. Using endothelial cells, we show that Cavin-2 is required for in vitro angiogenesis and also for endothelial cell proliferation, migration, and invasion. We noted a high level of Cavin-2 expression in the neovascular tufts in the mouse model of oxygen-induced retinopathy, suggesting a role for Cavin-2 in pathogenic angiogenesis. Interestingly, we also found that Cavin-2 regulates the production of nitric oxide (NO) in endothelial cells by controlling the stability and activity of the endothelial nitric-oxide synthase (eNOS) and that Cavin-2 knockdown cells produce much less NO than WT cells. Also, mass spectrometry, flow cytometry, and electron microscopy analyses indicated that Cavin-2 is secreted in endothelial microparticles (EMPs) and is required for EMP biogenesis. Taken together, our results indicate that in addition to its function in caveolae biogenesis, Cavin-2 plays a critical role in endothelial cell maintenance and function by regulating eNOS activity.

Highlights

Angiogenesis is a highly regulated process for formation of new blood vessels from pre-existing ones
To identify the novel genes involved in angiogenesis, we screened a list of candidate genes from the Human Protein Atlas (HPA) and BioGPS
We initially checked the protein expression levels of Cavin-2 in a panel of endothelial cells; we found that human aortic endothelial cells (HAEC), human umbilical vein endothelial cells (HUVECs), human pulmonary microvascular endothelial cells (HPMEC), and human retinal microvascular endothelial cells (HRMVEC) have high level of expression of Cavin-2 (Fig. 2B and supplemental Fig. S1)

Summary

Results

To identify the novel genes involved in angiogenesis, we screened a list of candidate genes from the Human Protein Atlas (HPA) and BioGPS. The number of branches and the endothelial tubular network were significantly reduced after silencing Cavin-2 in HAEC (Fig. 4B, p ϭ 0.022 and p ϭ 0.009, respectively), HUVEC (Fig. 4D, p ϭ 0.031 and p ϭ 0.025, respectively), HPMEC (Fig. Cavin-2 gene-specific morpholinos (sdpra-mo and sdpra-mo2) or control morpholinos. We found that the total protein levels of eNOS were decreased upon Cavin-2 knockdown in HUVECs cultured in complete endothelial cell growth medium (EGM-2) (Fig. 6A). There is more than a 4-fold (0.804 Ϯ 0.015 versus 0.182 Ϯ 0.061) decrease in total eNOS levels in Cavin-2 knockdown cells when compared with the control (Fig. 6D). These experiments clearly show that the loss of Cavin-2 significantly decreases the activity and stability of eNOS in HUVECs

D Cavin-2 β-Actin

Discussion

Experimental procedures