BACKGROUND: Systemic sclerosis (SSc) is characterized by endothelial cell (EC) apoptosis, impaired angiogenesis and peripheral microvasculopathy. Soluble α-Klotho (sKl) is a pleiotropic molecule with multiple effects on ECs, including antioxidant and vasculoprotective activities. On the EC surface, sKl interacts with vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) and transient receptor potential canonical-1 (TRPC-1) cation channel to control EC homeostasis. Here, we investigated whether sKl might act as a protective factor to improve angiogenesis in dermal microvascular endothelial cells (MVECs) from SSc patients (SSc-MVECs). METHODS: Wound healing assay was performed on healthy dermal MVECs (H-MVECs) challenged with sera from healthy controls or SSc patients with or without the addition of sKl. Capillary morphogenesis on Matrigel was assessed in H-MVECs and SSc-MVECs at basal conditions and treated with sKl, as well as in H-MVECs challenged with healthy or SSc sera in presence or absence of sKl. The expression of α-Klotho, VEGF165b, VEGFR-2, TRPC-1, Ki67 and active caspase-3 in H-MVECs and SSc-MVECs was investigated by western blotting. Immunostaining for α-Klotho was performed in H-MVECs and SSc-MVECs, and in healthy and SSc skin sections. RESULTS: Treatment with sKl effectively counteracted the inihibitory effects of SSc sera on wound healing ability and angiogenic performance of H-MVECs. The addition of sKl significantly improved angiogenesis and maintained over time capillary-like tube formation in vitro by SSc-MVECs. Stimulation of SSc-MVECs with sKl resulted in the upregulation of the proliferation marker Ki67 in parallel with the downregulation of proapoptotic active caspase-3. The expression of α-Klotho was significantly lower in SSc-MVECs than in H-MVECs. The expression of TRPC-1 was also significantly decreased, while that of VEGFR-2 and VEGF165b was significantly increased, in SSc-MVECs compared with H-MVECs. Challenge with sKl either significantly increased TRPC-1 or decreased VEGF165b in SSc-MVECs. Ex vivo analyses revealed that α-Klotho immunostaining was almost absent in the dermal microvascular network of SSc skin compared with control skin. CONCLUSIONS: Our findings provide the first evidence that α-Klotho is significantly decreased in the microvasculature in SSc skin and that sKl administration may effectively improve SSc-MVEC functions in vitro by acting as a powerful proangiogenic factor.

Proangiogenic effects of soluble α-Klotho on systemic sclerosis dermal microvascular endothelial cells / Mazzotta, Celestina; Manetti, Mirko; Rosa, Irene; Romano, Eloisa; Blagojevic, Jelena; BELLANDO RANDONE, Silvia; Bruni, Cosimo; Lepri, Gemma; Guiducci, Serena; Ibba, Lidia; MATUCCI CERINIC, Marco. - In: ARTHRITIS RESEARCH & THERAPY. - ISSN 1478-6354. - 19:(2017), pp. 27-27. [10.1186/s13075-017-1233-0]

Proangiogenic effects of soluble α-Klotho on systemic sclerosis dermal microvascular endothelial cells

MATUCCI CERINIC, MARCO
2017-01-01

Abstract

BACKGROUND: Systemic sclerosis (SSc) is characterized by endothelial cell (EC) apoptosis, impaired angiogenesis and peripheral microvasculopathy. Soluble α-Klotho (sKl) is a pleiotropic molecule with multiple effects on ECs, including antioxidant and vasculoprotective activities. On the EC surface, sKl interacts with vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) and transient receptor potential canonical-1 (TRPC-1) cation channel to control EC homeostasis. Here, we investigated whether sKl might act as a protective factor to improve angiogenesis in dermal microvascular endothelial cells (MVECs) from SSc patients (SSc-MVECs). METHODS: Wound healing assay was performed on healthy dermal MVECs (H-MVECs) challenged with sera from healthy controls or SSc patients with or without the addition of sKl. Capillary morphogenesis on Matrigel was assessed in H-MVECs and SSc-MVECs at basal conditions and treated with sKl, as well as in H-MVECs challenged with healthy or SSc sera in presence or absence of sKl. The expression of α-Klotho, VEGF165b, VEGFR-2, TRPC-1, Ki67 and active caspase-3 in H-MVECs and SSc-MVECs was investigated by western blotting. Immunostaining for α-Klotho was performed in H-MVECs and SSc-MVECs, and in healthy and SSc skin sections. RESULTS: Treatment with sKl effectively counteracted the inihibitory effects of SSc sera on wound healing ability and angiogenic performance of H-MVECs. The addition of sKl significantly improved angiogenesis and maintained over time capillary-like tube formation in vitro by SSc-MVECs. Stimulation of SSc-MVECs with sKl resulted in the upregulation of the proliferation marker Ki67 in parallel with the downregulation of proapoptotic active caspase-3. The expression of α-Klotho was significantly lower in SSc-MVECs than in H-MVECs. The expression of TRPC-1 was also significantly decreased, while that of VEGFR-2 and VEGF165b was significantly increased, in SSc-MVECs compared with H-MVECs. Challenge with sKl either significantly increased TRPC-1 or decreased VEGF165b in SSc-MVECs. Ex vivo analyses revealed that α-Klotho immunostaining was almost absent in the dermal microvascular network of SSc skin compared with control skin. CONCLUSIONS: Our findings provide the first evidence that α-Klotho is significantly decreased in the microvasculature in SSc skin and that sKl administration may effectively improve SSc-MVEC functions in vitro by acting as a powerful proangiogenic factor.
2017
Angiogenesis
Endothelial cells
Systemic sclerosis
α-Klotho
Immunology and Allergy
Rheumatology
Immunology
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11768/154514
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