Altered Properties of Endothelial Cells and Mesenchymal Stem Cells Underlying the Development of Scleroderma-like Vasculopathy in KLF5^+/- ;Fli-1^+/- Mice.

Abstract

In prevous studies, we established a new animal model, KLF5^+/- ;Fli-1^+/- mice, in which fundamental pathologic features of systemic sclerosis (SSc) are broadly recapitulated. SSc vasculopathy is believed to occur as a result of impaired vascular remodeling, but its detailed mechanism of action remains unknown. To address this, the present study investigated the properties of dermal microvascular endothelial cells (DMECs), bone marrow-derived endothelial progenitor cells (BM-EPCs), and bone marrow-derived mesenchymal stem cells (BM-MSCs), a precursor of pericytes, in KLF5^+/- ;Fli-1^+/- mice. Neovascularization and angiogenesis were assessed in KLF5^+/- ;Fli-1^+/- mice by in vivo Matrigel plug assay and in vitro tube formation assay, respectively. The properties of mouse BM-EPCs and BM-MSCs were assessed with in vitro studies. Dermal vasculature was visualized in vivo by injecting the mice with fluorescein isothiocyanate-conjugated dextran. Neovascularization was diminished in skin-embedded Matrigel plugs from KLF5^+/- ;Fli-1^+/- mice. DMECs from KLF5^+/- ;Fli-1^+/- mice showed defective tubulogenic activity, decreased expression of VE-cadherin and CD31, and an imbalance in the expression of Notch1/Dll4, suggesting that angiogenesis and anastomosis are disturbed. KLF5^+/- ;Fli-1^+/- mouse BM-MSCs exhibited enhanced proliferation and migration and increased collagen production following stimulation with transforming growth factor β1, indicating that these cells differentiate preferentially into myofibroblasts rather than pericytes. KLF5^+/- ;Fli-1^+/- mouse BM-EPCs displayed a transition toward mesenchymal cells, suggesting that vasculogenesis is impaired. Wound healing was delayed in KLF5^+/- ;Fli-1^+/- mice (mean ± SD healing time 15.67 ± 0.82 days versus 13.50 ± 0.84 days; P = 0.0017), and the vascular network was poorly developed in wound scar tissue. The characteristics observed in the KLF5^+/- ;Fli-1^+/- mouse model - specifically, impaired neovascularization and vascular maturation - are similar to those observed in human SSc, and could be at least partially attributable to the induction of SSc-like properties in DMECs, BM-EPCs, and BM-MSCs. These findings indicate the critical contribution of Klf5 and Fli1 deficiency in vascular cells and related cell precursors to the development of SSc vasculopathy.