CD47 Activation by Thrombospondin-1 in Lymphatic Endothelial Cells Suppresses Lymphangiogenesis and Promotes Atherosclerosis.

BACKGROUND: TSP1 (thrombospondin-1)-a well-known angiogenesis inhibitor-mediates differential effects via interacting with cell surface receptors including CD36 (cluster of differentiation) and CD47. However, the role of TSP1 in regulating lymphangiogenesis is not clear. Our previous study suggested the importance of cell-specific CD47 blockade in limiting atherosclerosis. Further, our experiments revealed CD47 as a dominant TSP1 receptor in lymphatic endothelial cells (LECs). As the lymphatic vasculature is functionally linked to atherosclerosis, we aimed to investigate the effects of LEC TSP1-CD47 signaling inhibition on lymphangiogenesis and atherosclerosis. METHODS: Murine atherosclerotic and nonatherosclerotic arteries were utilized to investigate TSP1 expression using Western blotting and immunostaining. LEC-specific knockout mice were used to determine the in vivo role of LEC Cd47 in lymphangiogenesis and atherosclerosis. Various in vitro cell-based assays, in vivo Matrigel plug implantation, molecular biological techniques, and immunohistological approaches were used to evaluate the underlying signaling mechanisms. RESULTS: Elevated TSP1 expression was observed in mouse atherosclerotic aortic tissue compared with nonatherosclerotic control tissue. TSP1 at pathological concentrations suppressed both in vitro and in vivo lymphangiogenesis. Mechanistically, TSP1 inhibited VEGF (vascular endothelial growth factor)-C-induced AKT and eNOS activation in LEC and attenuated NO (nitric oxide) production. Further, CD47 silencing in LEC prevented the effects of TSP1 on lymphangiogenic AKT-eNOS signaling and lymphangiogenesis. Atheroprone AAV (adeno-associated virus) 8-PCSK9-injected LEC-specific Cd47 knockout mice (Cd47ΔLEC) had reduced atherosclerosis in both aorta and aortic root compared with control mice (Cd47ΔWT). However, no differences in metabolic parameters including body weight, plasma total cholesterol levels, and fasting blood glucose were observed. Additional immunostaining experiments performed on aortic root cross-sections indicated higher lymphatic vessel density in Cd47ΔLEC mice in comparison to controls. CONCLUSIONS: These findings demonstrate that TSP1 inhibits lymphangiogenesis via activation of CD47 in LEC, and loss of LEC Cd47 attenuates atherosclerotic lesion formation. Collectively, these results identify LEC CD47 as a potential therapeutic target in atherosclerosis.

Immune modulatory effects of lenalidomide on the cultured peripheral blood mononuclear cells from vitiligo patients.

Vitiligo is a depigmentary disease in which epidermal melanocytes are lost. It is considered to be an autoimmune disease. Lenalidomide, an immunomodulatory drug is being employed in the treatment of various autoimmune and inflammatory disorders. In the present manuscript, the effect of lenalidomide on T cells and major cytokines in the cultured peripheral blood mononuclear cells (PBMCs) derived from vitiligo patients was checked. Eight patients with a clinical diagnosis of active vitiligo volunteered for the study. Blood was collected from them and PBMCs were isolated, cultured, and treated with lenalidomide. After 72 hours, PBMCs were harvested and checked for CD8+ and CD4+ T cells by flow cytometry. Further supernatant was collected and the levels of cytokines namely tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin-10 (IL-10), and interleukin-4 (IL-4) were checked using ELISA kits. Lenalidomide nonsignificantly decreased the level of CD8+ T cells but increased CD4+ T cells leading to increased CD4+ /CD8+ T cell ratio. It declined the level of pro-inflammatory cytokines, that is, TNF-α and IFN-γ whereas elevated anti-inflammatory cytokines, that is, IL-10 and IL-4, thus ultimately decreasing the ratio of pro-inflammatory to anti-inflammatory cytokines. Lenalidomide suppressed the proliferation of T lymphocytes and modulated the cytokines secretion toward an anti-inflammatory profile.

Differential expression of extracellular matrix proteins in the lesional skin of vitiligo patients.

Skin pigmentation is regulated by intricate interaction of the dermis and epidermis. The extracellular components present in the dermis play a very important role in the maintenance of skin homeostasis. Therefore, our objective was to check the expression of various ECM components secreted by the dermal fibroblasts in the lesional skin and non-lesional skin of vitiligo patients. For this study, skin punch biopsies (4 mm) were collected from lesional skin (n = 12), non-lesional skin (n = 6) of non-segmental vitiligo patient's (NSV) and healthy control skin (n = 10). Masson's trichrome staining was performed to check the collagen fibre. The expression of collagen type 1, IV, elastin, fibronectin, E-cadherin and integrin ß1 was checked by real-time PCR and immunohistochemistry. In this study, we demonstrated an increased expression of collagen type 1 in the lesional skin of vitiligo patients. The expression of collagen type IV, fibronectin, elastin and adhesion components such as E-cadherin and integrin ß1 was observed to be significantly decreased in the lesional skin of NSV patients as compared to healthy control, whereas insignificant difference was observed between non-lesional and control skin. Increased expression of collagen type 1 in the lesional skin of vitiligo patients might be inhibiting the migration of melanocytes, whereas the decreased expression of elastin, collagen type IV, fibronectin, E-cadherins and integrins in the lesional skin may inhibit adhesion, migration, growth and differentiation of cells.

Matricellular proteins in atherosclerosis development.

The extracellular matrix (ECM) is an intricate network composed of various multi-domain macromolecules like collagen, proteoglycans, and fibronectin, etc., that form a structurally stable composite, contributing to the mechanical properties of tissue. However, matricellular proteins are non-structural, secretory extracellular matrix proteins, which modulate various cellular functions via interacting with cell surface receptors, proteases, hormones, and cell-matrix. They play essential roles in maintaining tissue homeostasis by regulating cell differentiation, proliferation, adhesion, migration, and several signal transduction pathways. Matricellular proteins display a broad functionality regulated by their multiple structural domains and their ability to interact with different extracellular substrates and/or cell surface receptors. The expression of these proteins is low in adults, however, gets upregulated following injuries, inflammation, and during tumor growth. The marked elevation in the expression of these proteins during atherosclerosis suggests a positive association between their expression and atherosclerotic lesion formation. The role of matricellular proteins in atherosclerosis development has remained an area of research interest in the last two decades and studies revealed these proteins as important players in governing vascular function, remodeling, and plaque formation. Despite extensive research, many aspects of the matrix protein biology in atherosclerosis are still unknown and future studies are required to investigate whether targeting pathways stimulated by these proteins represent viable therapeutic approaches for patients with atherosclerotic vascular diseases. This review summarizes the characteristics of distinct matricellular proteins, discusses the available literature on the involvement of matrix proteins in the pathogenesis of atherosclerosis and suggests new avenues for future research.

Lenalidomide Augments Differentiation of Cultured Hair Follicle Derived Melanocyte Stem Cells Into Functional Melanocytes.

INTRODUCTION: Melanocyte progenitors are embryonically derived from the neural crest and subsequently get localized in hair follicles and epidermis to provide hair and skin pigmentation. These progenitor cells in hair follicles repeatedly proliferate and differentiate to maintain pigmentation. Vitiligo, a pigmentary disorder, is associated with loss of melanocytes. Repigmentation of vitiligo lesions mainly depends upon the proliferation, migration and differentiation of melanocyte stem cells (MelSCs) into functional melanocytes. The current study is designed to check the efficacy of lenalidomide, an imide drug in the differentiation of MelSCs into functional melanocytes. OBJECTIVES: The aim of the study is to check the effect of lenalidomide in the proliferation, migration of cultured hair follicle derived melanocyte stem cells and their differentiation into functional melanocytes. METHODS: Primary culture of MelSCs was established from whisker hair of C57BL/6 mice. Proliferation and migration of cultured cells were done by MTT assay and Boyden's chamber migration assay, respectively. Effect of lenalidomide on the MelSCs differentiation was checked at gene level by qPCR and protein expression was checked by immunocytochemistry. RESULTS: A significant increase in the migration of MelSCs in comparison to control was also observed. Lenalidomide treatment significantly increased the expression of melanocyte specific genes in cultured MelSCs as compared to control. CONCLUSIONS: From the results we concluded that lenalidomide induce the proliferation and migration of MelSCs and accelerate the differentiation of MelSCs into functional melanocytes.

Hydrogen sulfide donor activates AKT-eNOS signaling and promotes lymphatic vessel formation.

The lymphatic network is pivotal for various physiological functions in the human body. Accumulated evidence supports the role of therapeutic lymphangiogenesis in the treatment of several pathologies. Endogenous gasotransmitter, hydrogen sulfide (H2S) has been extensively studied for its potential as a pro-angiogenic factor and vascular function modulator. However, the role of H2S in governing lymphatic vessel formation, and underlying molecular mechanisms are understudied. The present study was designed to investigate the effects of H2S donor sodium hydrogen sulfide (NaHS) on lymphatic vascularization and pro-angiogenic signaling pathways using both in vitro and in vivo approaches. In vitro dose-response experiments showed increased proliferation and tube formation by NaHS-treated human lymphatic endothelial cells (LECs) compared with control cells. Immunoblotting performed with LEC lysates prepared after time-course NaHS treatment demonstrated increased activation of ERK1/2, AKT and eNOS after 20 min of NaHS stimulation. Further, NaHS treatment induced nitric oxide production, reduced reactive oxygen species generation, and promoted cell cycle in LECs. Additional cell cycle analysis showed that NaHS treatment abrogates oxidized LDL-induced cell cycle arrest in LECs. The results of in vivo Matrigel plug assay revealed increased lymphatic vessel density in Matrigel plugs containing NaHS compared with control plugs, however, no significant differences in angiogenesis and immune cell infiltration were observed. Collectively, these findings suggest that H2S donor NaHS promotes lymphatic vessel formation both in vitro and in vivo and may be utilized to promote reparative lymphangiogenesis to alleviate lymphatic dysfunction-related disorders.

Immune-modulatory effects of lenalidomide inhibited the progression of lesions in a vitiligo mouse model.

Vitiligo, an autoimmune disorder, is associated with altered cytokine levels and T lymphocytes. Lenalidomide modulates immune system components by altering cytokine production and regulating T-cell stimulation. In this study, effect of lenalidomide was checked on the development of vitiligo lesions, level of various cytokines, and T lymphocytes in the mouse model. The vitiligo mouse model was developed by immunizing C57BL/6 mouse with anti-mouse tyrosine-related protein 2. Lenalidomide was orally given to mice daily, and the effect was observed on the development of vitiligo lesions. The level of T lymphocytes in blood was checked by flow cytometry. Serum cytokine levels were checked by enzyme-linked immunosorbent assay. Vitiligo lesions were found significantly smaller in lenalidomide-treated mice models. It significantly decreased the serum levels of IFN-γ, TNF-α, IL-1ß, and IL-6 but elevated the levels of IL-4 and IL-10. It non-significantly elevated CD4+ /CD8+ T-cell ratio. Lenalidomide had an inhibitory effect on the development of vitiligo lesions in mice models by suppressing the serum level of pro-inflammatory cytokines and increasing anti-inflammatory cytokine levels. It modulated the immune response in vitiligo mice models toward an anti-inflammatory profile suggesting its use in the management of vitiligo.

Structural and physicochemical properties of Rheum emodi mediated Mg(OH)2 nanoparticles and their antibacterial and cytotoxic potential.

In the present investigation, Rheum emodi roots extract mediated magnesium hydroxide nanoparticles [Mg(OH)2 NPs] through the bio-inspired experimental technique were synthesised. Mg(OH)2 NPs were characterised by using various characterisation techniques such as field emission scanning electron microscopy, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and ultraviolet-visible spectroscopy. The formation of Mg(OH)2 NPs was confirmed by X-ray diffraction. The structural analysis confirmed the hexagonal crystal symmetry of Mg(OH)2 NPs with space group P-3m1 and space group no. 164 using the Rietveld refinement technique. TEM micrographs illustrated the nano-size formation of Mg(OH)2 NPs of spherical shape and size ∼14.86 nm. With the aid of FTIR data, plant metabolites such as anthraquinones have been identified as a stabilising and reducing agent for the synthesis of biogenic Mg(OH)2 NPs. The synthesised Mg(OH)2 NPs showed antimicrobial and cytotoxic potential against Gram-negative and Gram-positive bacteria such as Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923) and MDA-MB-231 human breast cancer cell lines.

Biosynthesis of hematite nanoparticles using Rheum emodi and their antimicrobial and anticancerous effects in vitro.

The synthesis of magnetic Hematite nanoparticles (α-Fe2O3) via green route has been a long lasting challenge for the scientific and technological fascination of many researchers. In the present investigation, iron oxide nanoparticles (α-Fe2O3) were synthesized using Rheum emodi roots in a cost effective and ecofriendly method. Their physicochemical property orchestration involved techniques such as UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), Transmission electron microscopy (TEM), Field emission scanning electron microscopy (FESEM), Energy-dispersive X-ray (EDX), X-ray diffraction (XRD), Thermogravimetric Analysis (TGA), Vibrating sample magnetometer (VSM), and Atomic force microscopy (AFM). Through TEM, FESEM and AFM analysis, α-Fe2O3NPs were confirmed spherical in shape and the average diameter of particle is ~12 nm as depicted through TEM image. Thermal property was investigated by TGA. Magnetic behavior was observed in R. emodi mediated α-Fe2O3NPs by magnetic hysteresis measurements. FTIR analysis revealed the presence of anthraquinones in R. emodi roots extract which play the central role in stabilization of the α-Fe2O3NPs. Further, the crystalline nature of the nanoparticle sample was determined with XRD experiment and SAED fringes calculation. The crystal was also confirmed with Rietveld refinement of XRD profile fitted with R-3c model Additionally, magnetic interaction with bacterial cell wall showed antimicrobial property against Escherichia coli, Gram-negative and Staphylococcus aureus, Gram-positive species. The approach transcribed in this paper reveals a novel methodology that utilizes α-Fe2O3 NPs to initiate apoptosis and inhibition of cervical cancer cells.