Proangiogenic effects of soluble α-Klotho on systemic sclerosis dermal microvascular endothelial cells.
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.
Department of Experimental and Clinical Medicine, Division of Rheumatology, University of Florence, AOUC, Largo Brambilla 3, 50134, Florence, Italy. mazzottacelestina@gmail.com.
Celestina
C
Mazzotta
Department of Experimental and Clinical Medicine, Section of Anatomy and Histology, University of Florence, Largo Brambilla 3, 50134, Florence, Italy.
Mirko
M
Manetti
Department of Experimental and Clinical Medicine, Division of Rheumatology, University of Florence, AOUC, Largo Brambilla 3, 50134, Florence, Italy.|Department of Experimental and Clinical Medicine, Section of Anatomy and Histology, University of Florence, Largo Brambilla 3, 50134, Florence, Italy.
Irene
I
Rosa
Department of Experimental and Clinical Medicine, Division of Rheumatology, University of Florence, AOUC, Largo Brambilla 3, 50134, Florence, Italy.
Eloisa
E
Romano
Department of Experimental and Clinical Medicine, Division of Rheumatology, University of Florence, AOUC, Largo Brambilla 3, 50134, Florence, Italy.
Jelena
J
Blagojevic
Department of Experimental and Clinical Medicine, Division of Rheumatology, University of Florence, AOUC, Largo Brambilla 3, 50134, Florence, Italy.
Silvia
S
Bellando-Randone
Department of Experimental and Clinical Medicine, Division of Rheumatology, University of Florence, AOUC, Largo Brambilla 3, 50134, Florence, Italy.
Cosimo
C
Bruni
Department of Experimental and Clinical Medicine, Division of Rheumatology, University of Florence, AOUC, Largo Brambilla 3, 50134, Florence, Italy.
Gemma
G
Lepri
Department of Experimental and Clinical Medicine, Division of Rheumatology, University of Florence, AOUC, Largo Brambilla 3, 50134, Florence, Italy.
Serena
S
Guiducci
Department of Experimental and Clinical Medicine, Section of Anatomy and Histology, University of Florence, Largo Brambilla 3, 50134, Florence, Italy.
Lidia
L
Ibba-Manneschi
Department of Experimental and Clinical Medicine, Division of Rheumatology, University of Florence, AOUC, Largo Brambilla 3, 50134, Florence, Italy.
Marco
M
Matucci-Cerinic
D000328:Adult; D000368:Aged; D015153:Blotting, Western; D042783:Endothelial Cells / Q000378:metabolism / Q000473:pathology*; D005260:Female; D005966:Glucuronidase / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D000090265:Klotho Proteins; D008297:Male; D055806:Microvessels / Q000378:metabolism / Q000473:pathology; D008875:Middle Aged; D018919:Neovascularization, Physiologic / Q000187:drug effects; D012595:Scleroderma, Systemic / Q000473:pathology*; D012867:Skin / Q000098:blood supply / Q000473:pathology
D005966:Glucuronidase; D000090265:Klotho Proteins
Angiogenesis; Endothelial cells; Systemic sclerosis; α-Klotho