Inhibition Of Endothelial-mesenchymal Transition Research Articles

Nintedanib ameliorates experimental pulmonary arterial hypertension via inhibition of endothelial mesenchymal transition and smooth muscle cell proliferation

Neointimal lesion and medial wall thickness of pulmonary arteries (PAs) are common pathological findings in pulmonary arterial hypertension (PAH). Platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) signaling contribute to intimal and medial vascular remodeling in PAH. Nintedanib is a tyrosine kinase inhibitor whose targets include PDGF and FGF receptors. Although the beneficial effects of nintedanib were demonstrated for human idiopathic pulmonary fibrosis, its efficacy for PAH is still unclear. Thus, we hypothesized that nintedanib is a novel treatment for PAH to inhibit the progression of vascular remodeling in PAs. We evaluated the inhibitory effects of nintedanib both in endothelial mesenchymal transition (EndMT)-induced human pulmonary microvascular endothelial cells (HPMVECs) and human pulmonary arterial smooth muscle cells (HPASMCs) stimulated by growth factors. We also tested the effect of chronic nintedanib administration on a PAH rat model induced by Sugen5416 (a VEGF receptor inhibitor) combined with chronic hypoxia. Nintedanib was administered from weeks 3 to 5 after Sugen5416 injection, and we evaluated pulmonary hemodynamics and PAs pathology. Nintedanib attenuated the expression of mesenchymal markers in EndMT-induced HPMVECs and HPASMCs proliferation. Phosphorylation of PDGF and FGF receptors was augmented in both intimal and medial lesions of PAs. Nintedanib blocked these phosphorylation, improved hemodynamics and reduced vascular remodeling involving neointimal lesions and medial wall thickening in PAs. Additionally, expressions Twist1, transcription factors associated with EndMT, in lung tissue was significantly reduced by nintedanib. These results suggest that nintedanib may be a novel treatment for PAH with anti-vascular remodeling effects.

Nicotinamide inhibits corneal endothelial mesenchymal transition and accelerates wound healing

Corneal endothelial cells (CECs) maintain the clarity of the cornea through the barrier and pump function. Ex vivo culture or injury may cause corneal endothelial-mesenchymal transition (EnMT) and lead to loss of function. In this study, we explored the effects of nicotinamide (NIC) on the wound healing of rabbit corneal endothelium and the proliferation, migration, and EnMT of cultured human CEC lines. The animal results showed that corneal clarity was rapidly recovered within seven days through topical application of NIC in the rabbits with mechanical injury of the corneal endothelium, while the control corneas remained edematous and cloudy. Whole-mounted corneal staining found the expressions of Na+/K+-ATPase, aquaporin-1, and zonula occludens-1 were mainly localized to the boundaries of regenerated endothelium in NIC-treated eyes, in contrast to the scattered staining in vehicle-treated eyes. Interestingly, we found that NIC application inhibited the expression of typical EnMT marker alpha-smooth muscle actin, which appeared in the rabbit corneal endothelial wound healing. In vitro, NIC promoted the proliferation, but not the migration, of cultured human CECs. Moreover, NIC effectively inhibited transforming growth factor beta-1-induced corneal EnMT and decreased the levels of EnMT regulators snail and slug. Therefore, our study indicates that NIC enhances corneal endothelial wound healing through the promotion of proliferation and the inhibition of EnMT, which may provide a potential pharmaceutical agent for treating corneal endothelial dysfunction.

NLRP3 Inflammasome Activation Contributes to Mechanical Stretch–Induced Endothelial-Mesenchymal Transition and Pulmonary Fibrosis

Mechanical ventilation can induce lung fibrosis. This study aimed to investigate whether ventilator-induced lung fibrosis was associated with endothelial-mesenchymal transition and to uncover the underlying mechanisms. Randomized, controlled animal study and cell culture study. University research laboratory. Adult male Institute of Cancer Research, NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) knockout and wild-type mice. Primary cultured mouse lung vascular endothelial cells. Institute of Cancer Research, NLRP3 knockout and wild-type mice were subjected to mechanical ventilation (20 mL/kg) for 2 hours. Mouse lung vascular endothelial cells were subjected to cyclic stretch for 24 hours. Mice subjected to mechanical ventilation exhibited increases in collagen deposition, hydroxyproline and type I collagen contents, and transforming growth factor-β1 in lung tissues. Ventilation-induced lung fibrosis was associated with increased expression of mesenchymal markers (α smooth muscle actin and vimentin), as well as decreased expression of endothelial markers (vascular endothelial-cadherin and CD31). Double immunofluorescence staining showed the colocalization of CD31/α smooth muscle actin, CD31/vimentin, and CD31/fibroblast-specific protein-1 in lung tissues, indicating endothelial-mesenchymal transition formation. Mechanical ventilation also induced NLRP3 inflammasome activation in lung tissues. In vitro direct mechanical stretch of primary mouse lung vascular endothelial cells resulted in similar NLRP3 activation and endothelial-mesenchymal transition formation, which were prevented by NLRP3 knockdown. Furthermore, mechanical stretch-induced endothelial-mesenchymal transition and pulmonary fibrosis were ameliorated in NLRP3-deficient mice as compared to wild-type littermates. Mechanical stretch may promote endothelial-mesenchymal transition and pulmonary fibrosis through a NLRP3-dependent pathway. The inhibition of endothelial-mesenchymal transition by NLRP3 inactivation may be a viable therapeutic strategy against pulmonary fibrosis associated with mechanical ventilation.

Cinnamaldehyde attenuates pressure overload-induced cardiac fibrosis via inhibition of endothelial mesenchymal transition

Objective: The aim of this study is to investigate the underlying mechanism of cinnamaldehyde attenuating pressure overload-induced cardiac fibrosis. Methods: The mice were randomly divided into control group, model group and treatment group by random number table and each group had 8 mice.Cardiac hypertrophy was induced by aortic banding. Heart vascular density was detected by immunohistochemical staining of CD31.The expression level of stromal cells marker α-smooth muscle actin (α-SMA) was detected by immunofluorescence staining in different groups.The expression levels of endothelial cell associated markers and stromal cell associated markers were detected by using Western blotting.The possible molecular pathway was also screened by using Western blotting. Human umbilical vein endothelial cell (HUVECs) were stimulated with TGFβ1 and cultured with 10 nmol/L cinnamomum for 24 hour to further confirm the mechanism. Results: Eight weeks after operation, the vascular density was significantly decreased in model group mice heart.The expressions of stromal cells markers were increased (α-SMA: 2.57±0.38; Vimentin: 0.58±0.02) and endothelial cell markers were reduced (CD31: 0.58±0.29; CD34: 0.62±0.21). While cinnamicaldehyde treatment significantly increased the mouse heart vascular density, increased endothelial cell markers expression (CD31: 1.51±0.11; CD34: 2.37±0.44; P<0.05), and reduced stromal cells marker expression (α-SMA: 1.22±0.14; Vimentin: 0.35±0.03; P<0.05). Further studies showed that the anti-fibrosis effect of cinnamicaldehyde was mainly through the TGFβ /smad signaling pathway.10 nmol/L cinnamomum attenuated TGFβ1 induced endothelial mesenchymal transition in HUVECs. Conclusion: Cinnamaldehyde may be able to retard the progression of cardiac fibrosis, via blocking endothelial to mesenchymal transition, which, in verse, is through regulating TGFβ /smad signaling pathway.

Role of Kallistatin Treatment in Aging and Cancer by Modulating miR-34a and miR-21 Expression.

Kallistatin is an endogenous protein that regulates differential signaling pathways and a wide spectrum of biological activities via its two structural elements: an active site and a heparin-binding domain. Kallistatin via its heparin-binding site inhibits vascular inflammation and oxidative stress by antagonizing TNF-α-induced NADPH oxidase activity, NF-κB activation, and inflammatory gene expression in endothelial cells. Moreover, kallistatin via its active site inhibits microRNA-34a (miR-34a) synthesis and stimulates eNOS and SIRT1 expression in endothelial progenitor cells, whereas its heparin-binding site is crucial for blocking TNF-α-induced miR-21 expression and oxidative stress, thus reducing cellular senescence. By downregulating miR-34a and miR-21 expression, kallistatin treatment attenuates oxidative damage and aortic senescence in streptozotocin-induced diabetic mice and extends Caenorhabditis elegans lifespan under stress conditions. Likewise, kallistatin through the heparin-binding site inhibits TGF-β-induced miR-21 synthesis and oxidative stress in endothelial cells, resulting in inhibition of endothelial-mesenchymal transition, a process contributing to fibrosis and cancer. Furthermore, kallistatin's active site is essential for stimulating miR-34a and p53 expression and inhibiting the miR-21-Akt-Bcl-2 signaling pathway, thus inducing apoptosis in breast cancer cells. These findings reveal novel mechanisms of kallistatin in protection against senescence, aging, and cancer development by modulating miR-34a and miR-21 levels and inhibiting oxidative stress.

MiR-630 Inhibits Endothelial-Mesenchymal Transition by Targeting Slug in Traumatic Heterotopic Ossification.

Heterotopic ossification (HO) is the abnormal formation of mature bone in extraskeletal soft tissues that occurs as a result of inflammation caused by traumatic injury or associated with genetic mutation. Despite extensive research to identify the source of osteogenic progenitors, the cellular origins of HO are controversial and the underlying mechanisms, which are important for the early detection of HO, remain unclear. Here, we used in vitro and in vivo models of BMP4 and TGF-β2-induced HO to identify the cellular origin and the mechanisms mediating the formation of ectopic bone in traumatic HO. Our results suggest an endothelial origin of ectopic bone in early phase of traumatic HO and indicate that the inhibition of endothelial-mesenchymal transition by miR-630 targeting Slug plays a role in the formation of ectopic bone in HO. A matched case-control study showed that miR-630 is specifically downregulated during the early stages of HO and can be used to distinguish HO from other processes leading to bone formation. Our findings suggest a potential mechanism of post-traumatic ectopic bone formation and identify miR-630 as a potential early indicator of HO.

Abstract A111: Plant galactolipid dLGG suppresses lung metastasis of melanoma through modulating endothelial-mesenchymal transition extravasation and oxylipins dynamics

Abstract A galactolipid 1,2-di-O-linolenoyl-3-O-ß-galactopyranosyl-sn-glycerol (designated dLGG) isolated from medicinal plant Crassocephalum rabens (Asteraceae) was demonstrated to be anti-inflammatory and anti-melanoma tumor growth in syngeneic mouse system in our laboratory. In this study, an experimental lung metastasis mouse model using a B16 melanoma cell clone (designated B16COX-2/Luc) carrying COX-2 promoter driven luciferase reporter gene was established to monitor the cancer cell growth and metastasis in animals and the dLGG effect. Our data showed that dLGG (20 mg/kg BW, dLGG−20), cisplatin (2 mg/kg BW, CP−2), and combination drug treatment (CP−2+dLGG−20) significantly inhibited lung metastasis of melanoma in mice from 100% at tumor control group to 9.2%, 43.1%, and 4.1%, respectively, as determined by bioluminescence intensity. The dLGG (30±8 days), cisplatin (35±9 days) and combination treatment group (29±7 days) mice showed longer mean survival time than tumor control group (22±1 days)(P&amp;lt;0.02). Immunohistochemistry study on lung tissues showed that dLGG and CP+dLGG combination treatment inhibited the expression of cell proliferation (PCNA), cell invasion (MMP2 and MMP9), and cell metastasis (COX-2, CXCR4 and CD9) markers, but less effective in cisplatin group. We investigated the underlying mechanism of dLGG on inhibiting melanoma cell activity, including endothelial-mesenchymal transition (EMT), and extravasation through tight junction permeability. Our data showed that EMT markers expression, such as effector vimentin, regulator snail, and inducer TGF-ß, ß-catenin and TNF-α were down-regulated in dLGG-treated melanoma cells. Melanoma culture medium (CM) induced tight junction permeability in HUVECs monolayer which was attenuated by dLGG treatment. Furthermore, VEGF-induced melanoma cell extravasation via activation of FAK/PI3K/AKT pathway could be also inhibited by dLGG treatment. Oxylipins, a specific group of lipid mediators, play a crucial role in inflammation and are also implied in cancer and other diseases. We have established oxylipin metabolomics platform using UPLC/triple-quadrupole mass spectrometry coupled with partial least squares discriminant analysis (PLS-DA), to investigate the role of oxylipins involved in melanoma lung metastasis mice. The results of serum oxylipins analysis showed that cisplatin treatment not only induced significantly on anti-inflammatory 11,12-EET, but also increased proinflammatory 11-HETE and LTB4 (10- to 20-fold), relative to the tumor control. dLGG alone or dLGG and cisplatin combination treatment increased the anti-inflammatory EPA, EETs, 15-HEPE and 17-HDOHE (2- to 13-fold) in mouse serum. This study sheds light on the novel activity and the underlying molecular mechanisms of phytoagent dLGG on inhibition of melanoma cell EMT, tight junction permeability, and extravasation that support in part the in vivo therapeutic effect of dLGG on melanoma lung metastasis. Citation Format: Chung-Chih Yang, Meng-Ting Chang, Lie-Fen Shyur. Plant galactolipid dLGG suppresses lung metastasis of melanoma through modulating endothelial-mesenchymal transition extravasation and oxylipins dynamics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A111.

Mesenchymal stem cell-derived microparticles ameliorate peritubular capillary rarefaction via inhibition of endothelial-mesenchymal transition and decrease tubulointerstitial fibrosis in unilateral ureteral obstruction.

IntroductionMicroparticles (MPs) derived from kidney-derived mesenchymal stem cells (KMSCs) have recently been reported to ameliorate rarefaction of peritubular capillaries (PTC) in ischemic kidneys via delivery of proangiogenic effectors. This study aimed to investigate whether KMSC-derived MPs show anti-fibrotic effects by ameliorating endothelial-to-mesenchymal transition (EndoMT) in human umbilical vein endothelial cells (HUVEC) in vitro and by preserving PTC in kidneys with unilateral ureteral obstruction (UUO) in vivo.MethodsMPs isolated from the supernatants of KMSC were co-cultured with HUVEC to assess their in vitro biologic effects on endothelial cells. Mice were treated with MPs via the tail vein after UUO injury to assess their anti-fibrotic and PTC sparing effects. Renal tubulointerstitial damage and inflammatory cell infiltration were examined with Masson’s trichrome, F4/80 and α-smooth muscle actin (α-SMA) staining and PTC rarefaction index was determined by CD31 staining.ResultsKMSC-derived MPs significantly ameliorated EndoMT and improved in vitro proliferation of TGF-β1 treated HUVEC. In vivo administration of KMSC-derived MPs significantly inhibited EndoMT of PTC endothelial cells and improved PTC rarefaction in UUO kidneys. Furthermore, administration of KMSC-derived MPs inhibited inflammatory cell infiltration as well as tubulointerstitial fibrosis in UUO mice as demonstrated by decreased F4/80 and α-SMA-positive cells and Masson’s trichrome staining, respectively.ConclusionsOur results suggest that KMSC-derived MPs ameliorate PTC rarefaction via inhibition of EndoMT and protect against progression of renal damage by inhibiting tubulointerstitial fibrosis.

Epidermal growth factor receptor signaling and the progression of diabetic nephropathy.

Epidermal growth factor receptor signaling and the progression of diabetic nephropathy.

Response to Hepatocyte Growth Factor and Cardiomyopathy in Dialysis Patients

Hepatocyte growth factor (HGF) is a mesenchyme-derived pleiotropic factor that regulates cell growth, cell motility, and morphogenesis of various types of cells and is, thus, considered a humoral mediator of epithelial-mesenchymal interactions. Although HGF was originally identified as a potent mitogen for hepatocytes, HGF is now well known as an angiogenic growth factor. Because the presence of its specific receptor, c-met, is observed in vascular cells and cardiac myocytes, the cardiovascular HGF-met system has been a center of interest. Malatino et al1 suggested that an increase in circulating HGF might be compensated to myocardial damage in dialysis patients, because our recent report demonstrated that HGF reduced cardiac fibrosis through the inhibition of endothelial mesenchymal transition and the transformation of fibroblasts to myofibroblasts.2 We agree with their hypothesis that circulating HGF might be increased to attenuate the cardiovascular damage. In various pathophysiological conditions, such as hypertension, peripheral …