Abstract
In systemic sclerosis (SSc), the possible involvement of lymphatic microcirculation and lymphangiogenesis has traditionally been overshadowed by the greater emphasis placed on dysfunctional blood vascular system and angiogenesis. In the present in vitro study, we explore for the first time whether the SSc microenvironment may interfere with lymphangiogenesis, a complex, multi-step process in which lymphatic microvascular endothelial cells (LMVECs) sprout, migrate, and proliferate to generate new lymphatic capillaries. Normal human adult dermal LMVECs from three donors were treated with serum from SSc patients (n = 8), serum from healthy individuals (n = 8), or recombinant human vascular endothelial growth factor (VEGF)-C as a positive control for lymphangiogenesis. Cell proliferation, Boyden chamber Matrigel chemoinvasion, wound healing capacity, and lymphatic capillary morphogenesis on Geltrex were assayed. VEGF-C serum levels were measured by enzyme-linked immunosorbent assay. Gene and protein expression levels of the lymphangiogenic orchestrators VEGF receptor-3 (VEGFR-3)/Flt-4 and neuropilin-2 (NRP-2) were determined by real-time PCR and Western blotting, respectively. Conditioning with SSc serum significantly inhibited LMVEC proliferation, Matrigel invasion, and wound healing capacity with respect to healthy serum. The ability of LMVECs to form lymphatic tubes on Geltrex was also severely compromised in the presence of SSc serum. VEGF-C levels were comparable in SSc and healthy sera. Treatment with SSc serum resulted in a significant downregulation of both VEGFR-3/Flt-4 and NRP-2 mRNA and protein levels. In SSc, the pathologic environment severely hampers every lymphangiogenesis step, likely through the reduction of pro-lymphangiogenic VEGFR-3/NRP-2 co-receptor signaling. The impairment of the lymphangiogenic process opens a new scenario underlying SSc vascular pathophysiology, which is worth investigating further.
Highlights
The pathogenic scenario of systemic sclerosis (SSc, scleroderma) is dominated by peripheral microvascular damage and immune system disturbances, culminating in progressive fibrosis of the skin and internal organs [1,2]
Previous studies have extensively demonstrated that treatment with SSc serum is capable of severely impairing the angiogenic performance of dermal blood microvascular endothelial cells, and that in SSc, dysfunctional angiogenesis may make a substantial contribution to the failure of pIenrt.ipJ.hMeorla
To shed some light on the possible mechanisms underlying the anti-lymphangiogenic effect exerted by SSc serum on lymphatic microvascular endothelial cells (LMVECs), we investigated whether it could induce any relevant changes in the expression of cell surface receptors, which act as important mediators of the pro-lymphangiogenic activity of vascular endothelial growth factor (VEGF)-C
Summary
The pathogenic scenario of systemic sclerosis (SSc, scleroderma) is dominated by peripheral microvascular damage and immune system disturbances, culminating in progressive fibrosis of the skin and internal organs [1,2]. A few clinical and histopathologic studies have demonstrated that clinically-affected SSc skin displays lymphatic microvascular abnormalities, including a severe reduction in both superficial and deep dermal microlymphatic networks that has been correlated with the presence of fingertip ulcers and linked to cutaneous disease progression from an early edematous stage toward overt fibrotic remodeling [9,10,11,12]. It has been suggested that dermal lymphatic microangiopathy may take place as a consequence of blood capillary leaking in very early SSc, when “puffy fingers” are a clinical hallmark [5]. Dermal blood capillary leaks result in greater amounts of fluid and macromolecules in the interstitium, followed by maximal increase in lymph flow, which provokes microlymphatic vessel overload/insufficiency and consequent tissue accumulation of protein-rich interstitial fluid, immune cells, chemokines, and growth factors, clinically manifesting as skin edema [5]. The objective of our in vitro study was to investigate, for the first time, whether functional defects in the lymphangiogenic process may be part of the complex scenario underlying SSc peripheral microvasculopathy
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