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

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.

Prognostic and diagnostic significance of Cavin 2 in lung adenocarcinoma

“…Cavin 2, a serum deprivation protein response gene, is enriched in lung tissue [12]. In this study, our data showed that low Cavin 2 expression was more easily observed in lung adenocarcinoma compared with normal lung tissues, indicating that Cavin 2 might be a tumor suppressor gene and Cavin 2 depletion might be implicated in the formation of lung adenocarcinoma.…”

“…Unozawa et al reported that Cavin 2 up-regulation slowed cellular proliferation by regulating cell-cycle arrest in oral cancer [18]. Boopathy et al reported that Cavin 2 was required for endothelial cell proliferation, invasion and migration [12]. However, the effect of Cavin 2 expression on cell differentiation, proliferation, migration and invasion is still unclear in lung adenocarcinoma.…”

“…Cavin 2, a membrane-bound phosphatidylserine-binding protein, is regarded as a critical organizer of caveolae and a regulator of angiogenesis [12,13]. Cavin 2 is required for angiogenesis and plays a key role in cell proliferation, migration and invasion [14,15].…”

A Role of Agrin in Maintaining the Stability of Vascular Endothelial Growth Factor Receptor-2 during Tumor Angiogenesis

“…We anticipated a dramatic reduction in e-NO production as an outcome of decreased VEGFR2-e-NOS signaling in the absence of Agrin. Likewise, Agrin depletion severely hampered the production of e-NO, as measured by the formation of triazolo-fluorescein by NO indicator 4,5-diaminofluorescein diacetate (DAF-2-DA) (Boopathy et al, 2017;Govers et al, 2002) ( Figure 7F). The resulting inhibition of e-NO production and phospho-e-NOS status in Agrin-depleted HUVECs was comparable to that caused by treatment with N u -nitro-L-arginine-methyl ester hydrochloride (L-NAME), a known inhibitor for NO production (Boopathy et al, 2017) (Figure 7G).…”

“…Likewise, Agrin depletion severely hampered the production of e-NO, as measured by the formation of triazolo-fluorescein by NO indicator 4,5-diaminofluorescein diacetate (DAF-2-DA) (Boopathy et al, 2017;Govers et al, 2002) ( Figure 7F). The resulting inhibition of e-NO production and phospho-e-NOS status in Agrin-depleted HUVECs was comparable to that caused by treatment with N u -nitro-L-arginine-methyl ester hydrochloride (L-NAME), a known inhibitor for NO production (Boopathy et al, 2017) (Figure 7G). The restoration of tube formation induced by soluble Agrin was blocked by L-NAME treatment in Agrindepleted HUVECs, suggesting that soluble Agrin required e-NO production to induce in vitro angiogenesis ( Figure S6C).…”