Correction to: Concurrent vitamin D supplementation and exercise training improve cardiac fibrosis via TGF-β/Smad signaling in myocardial infarction model of rats

Journal of Physiology and Biochemistry - A Correction to this paper has been published: https://doi.org/10.1007/s13105-021-00795-z     

Quercetin prevents myocardial infarction adverse remodeling in rats by attenuating TGF-β1/Smad3 signaling: Different mechanisms of action

This study investigated the anti-remodeling and anti-fibrotic and effect of quercetin (QUR) in the remote non-infarcted of rats after myocardial infarction (MI). Rats were divided as control, control + QUR, MI, and MI + QUR. MI was introduced to the rats by ligating the eft anterior descending (LAD) coronary artery. All treatments were given for 30 days, daily. QUR persevered the LV hemodynamic parameters and prevented remote myocardium damage and fibrosis. Also, QUR supressed the generation of ROS, increased the nuclear levels of Nrf2, and enhanced SOD and GSH levels in the LVs of the control and MI model rats. It also reduced angiotensin II, nuclear level/activity of the nuclear factor NF-κβ p65, and protein expression of TGF-β1, α-SMA, and total/phospho-smad3 in the LVs of both groups. Concomitantly, QUR upregulated LV smad7 and BMP7. In conclusion, QUR prevents MI-induced LV remodeling by antioxidant, anti-inflammatory, and anti-fibroticα effects mediated by ROS scavenging, suppressing NF-κβ, and stimulating Nrf-2, Smad7, and BMP7.     

Protective effect of TRPV1 against renal fibrosis via inhibition of TGF-β/Smad signaling in DOCA-salt hypertension

To investigate the effects of the transient receptor potential vanilloid type 1 (TRPV1) channel on renal extracellular matrix (ECM) protein expression including collagen deposition and the transforming growth factor β (TGF-β)/Smad signaling pathway during salt-dependent hypertension, wild-type (WT) and TRPV1-null (TRPV1?/?) mutant mice were uninephrectomized and given deoxycorticosterone acetate (DOCA)-salt for 4 wks. TRPV1 gene ablation exaggerated DOCA-salt-induced impairment of renal function as evidenced by increased albumin excretion (μg/24 h) compared with WT mice (83.7 ± 7.1 versus 28.3 ± 4.8, P < 0.05), but had no apparent effect on mean arterial pressure (mmHg) as determined by radiotelemetry (141 ± 4 versus 138 ± 3, P > 0.05). Morphological analysis showed that DOCA-salt-induced glomerulosclerosis, tubular injury and macrophage infiltration (cells/mm2) were increased in TRPV1?/? compared with WT mice (0.74 ± 0.08 versus 0.34 ± 0.04; 3.14 ± 0.26 versus 2.00 ± 0.31; 68 ± 5 versus 40 ± 4, P < 0.05). Immunostaining studies showed that DOCA-salt treatment decreased nephrin but increased collagen type I and IV as well as phosphorylated Smad2/3 staining in kidneys of TRPV1?/? compared with WT mice. Hydroxyproline assay and Western blot showed that DOCA-salt treatment increased collagen content (μg/mg dry tissue) and fibronectin protein expression (%β-actin arbitrary units) in the kidney of TRPV1?/? compared with WT mice (26.7 ± 2.7 versus 17.4 ± 1.8; 0.93 ± 0.07 versus 0.65 ± 0.08, P < 0.05). Acceleration of renal ECM protein deposition in DOCA-salt-treated TRPV1?/? mice was accompanied by increased TGF-β1, as well as phosphorylation of Smad2/3 protein expression (%β-actin arbitrary units) compared with DOCA-salt-treated WT mice (0.61 ± 0.07 versus 0.32 ± 0.05; 0.57 ± 0.07 versus 0.25 ± 0.05; 0.71 ± 0.08 versus 0.40 ± 0.06, P < 0.05). These results show that exaggerated renal functional and structural injuries are accompanied by increased production of ECM protein and activation of the TGF-β/Smad2/3 signaling pathway. These data suggest that activation of TRPV1 attenuates the progression of renal fibrosis possibly via suppression of the TGF-β and its downstream regulatory signaling pathway.     

Dapagliflozin alleviates cardiac fibrosis through suppressing EndMT and fibroblast activation via AMPKα/TGF-β/Smad signalling in type 2 diabetic rats

Diabetic cardiomyopathy (DCM) is one of the leading causes of heart failure in patients with diabetes mellitus, with limited effective treatments. The cardioprotective effects of sodium-glucose cotransporter 2(SGLT2) inhibitors have been supported by amounts of clinical trials, which largely fills the gap. However, the underlying mechanism still needs to be further explored, especially in terms of its protection against cardiac fibrosis, a crucial pathophysiological process during the development of DCM. Besides, endothelial-to-mesenchymal transition (EndMT) has been reported to play a pivotal role in fibroblast multiplication and cardiac fibrosis. This study aimed to evaluate the effect of SGLT2 inhibitor dapagliflozin (DAPA) on DCM especially for cardiac fibrosis and explore the underlying mechanism. In vivo, the model of type 2 diabetic rats was built with high-fat feeding and streptozotocin injection. Untreated diabetic rats showed cardiac dysfunction, increased myocardial fibrosis and EndMT, which was attenuated after treatment with DAPA and metformin. In vitro, HUVECs and primary cardiac fibroblasts were treated with DAPA and exposed to high glucose (HG). HG-induced EndMT in HUVECs and collagen secretion of fibroblasts were markedly inhibited by DAPA. Up-regulation of TGF-β/Smad signalling and activity inhibition of AMPKα were also reversed by DAPA treatment. Then, AMPKα siRNA and compound C abrogated the anti-EndMT effects of DAPA in HUVECs. From above all, our study implied that DAPA can protect against DCM and myocardial fibrosis through suppressing fibroblast activation and EndMT via AMPKα-mediated inhibition of TGF-β/Smad signalling.     

Dynamics of TGF-β/Smad signaling

The physiological responses to TGF-β stimulation are diverse and vary amongst different cell types and environmental conditions. Even though the principal molecular components of the canonical and the non-canonical TGF-β signaling pathways have been largely identified, the mechanism that underlies the well-established context dependent physiological responses remains a mystery. Understanding how the components of TGF-β signaling function as a system and how this system functions in the context of the global cellular regulatory network requires a more quantitative and systematic approach. Here, we review the recent progress in understanding TGF-β biology using integration of mathematical modeling and quantitative experimental analysis. These studies reveal many interesting dynamics of TGF-β signaling and how cells quantitatively decode variable doses of TGF-β stimulation.     

Physalin D attenuates hepatic stellate cell activation and liver fibrosis by blocking TGF-β/Smad and YAP signaling

BackgroundHepatic fibrosis is considered integral to the progression of chronic liver diseases, as it leads to the development of cirrhosis and hepatocellular carcinoma. The activation of hepatic stellate cells (HSCs) is the dominant event in hepatic fibrogenesis. The transforming growth factor-β1 (TGF-β1) and Yes-associated protein (YAP) pathways play a pivotal role in HSC activation, hepatic fibrosis and cirrhosis progression. Therefore, targeting the TGF-β/Smad and YAP signaling pathways is a promising strategy for antifibrotic therapy.PurposeThe present study investigated the protective effects of Physalin D (PD), a withanolide isolated from Physalis species (Solanaceae), against liver fibrosis and further elucidated the mechanisms involved in vitro and in vivo.Study design/methodsWe conducted a series of experiments using carbon tetrachloride (CCl₄)- and bile duct ligation (BDL)-induced fibrotic mice and cultured LX-2 cells. Serum markers of liver injury, and the morphology, histology and fibrosis of liver tissue were investigated. Western blot assays and quantitative real-time PCR were used to investigate the mechanisms underlying the antifibrotic effects of PD.ResultPD decreased TGF-β1-induced COL1A1 promoter activity. PD inhibited TGF-β1-induced expression of Collagen I and α-smooth muscle actin (α-SMA) in human hepatic stellate LX-2 cells. PD significantly ameliorated hepatic injury, including transaminase activities, histology, collagen deposition and α-SMA, in CCl₄- or BDL-induced mice. Moreover, PD markedly decreased the expression of phosphorylated Smad2/3 in vitro and in vivo. Furthermore, PD significantly decreased YAP protein levels, and YAP knockdown did not further enhance the effects of PD, namely α-SMA inhibition, Collagen I expression and YAP target gene expression in LX-2 cells.ConclusionThese results clearly show that PD ameliorated experimental liver fibrosis by inhibiting the TGF-β/Smad and YAP signaling pathways, indicating that PD has the potential to effectively treat liver fibrosis.     

The Notch γ-secretase inhibitor ameliorates kidney fibrosis via inhibition of TGF-β/Smad2/3 signaling pathway activation

Kidney fibrosis is a common feature of chronic kidney disease (CKD). A recent study suggests that abnormal Notch signaling activation contributes to the development of renal fibrosis. However, the molecular mechanism that regulates this process remains unexplored. Unilateral ureteral obstruction (UUO) or sham-operated C57BL6 mice (aged 10 weeks) were randomly assigned to receive dibenzazepine (DBZ, 250 μg/100 g/d) or vehicle for 7 days. Histologic examinations were performed on the kidneys using Masson's trichrome staining and immunohistochemistry. Real-time PCR and western blot analysis were used for detection of mRNA expression and protein phosphorylation. The expression of Notch 1, 3, and 4, Notch intracellular domain (NICD), and its target genes Hes1 and HeyL were upregulated in UUO mice, while the increase in NICD protein was significantly attenuated by DBZ. After 7 days, the severity of renal fibrosis and expression of fibrotic markers, including collagen 1α1/3α1, fibronectin, and α-smooth muscle actin, were markedly increased in UUO compared with sham mice. In contrast, administration of DBZ markedly attenuated these effects. Furthermore, DBZ significantly inhibited UUO-induced expression of transforming growth factor (TGF)-β, phosphorylated Smad 2, and Smad 3. Mechanistically, Notch signaling activation in tubular epithelial cells enhanced fibroblast proliferation and activation in a coculture experiment. Our study provides evidence that Notch signaling is implicated in renal fibrogenesis. The Notch inhibitor DBZ can ameliorate this process via inhibition of the TGF-β/Smad2/3 signaling pathway, and might be a novel drug for preventing chronic kidney disease.     

Bushenhuoxue formula accelerates fracture healing via upregulation of TGF-β/Smad2 signaling in mesenchymal progenitor cells

BackgroundAlthough Bushenhuoxue formula (BSHXF) is successfully used as a non-traumatic therapy in treating bone fracture in China, the molecular mechanism underlying its effects remains poorly understood.PurposeThe present study aims to explore the therapeutic effects of BSHXF on fracture healing in mice and the underlying mechanism.MethodsWe performed unilateral open transverse tibial fracture procedure in C57BL/6 mice which were treated with or without BSHXF. Fracture callus tissues were collected and analyzed by X-ray, micro-CT, biomechanical testing, histopathology and quantitative gene expression analysis. Tibial fracture procedure was also performed in Cre-negative and Gli1-CreER; Tgfbr2flox/flox conditional knockout (KO) mice (Tgfbr2^Gli1ER) to determine if BSHXF enhances fracture healing in a TGF-β-dependent manner. In addition, scratch-wound assay and cell counting kit-8 (CCK-8) assay were used to evaluate the effect of BSHXF on cell migration and cell proliferation in C3H10T1/2 mesenchymal stem cells, respectively.ResultsBSHXF promoted endochondral ossification and enhanced bone strength in wild-type (WT) or Cre- control mice. In contrast, BSHXF failed to promote bone fracture healing in Tgfbr2^Gli1ER conditional KO mice. In the mice receiving BSHXF treatment, TGF-β/Smad2 signaling was significantly activated. Moreover, BSHXF enhanced cell migration and cell proliferation in C3H10T1/2 cells, which was strongly attenuated by the small molecule inhibitor SB525334 against TGF-β type I receptor.ConclusionThese data demonstrated that BSHXF promotes fracture healing by activating TGF-β/Smad2 signaling. BSHXF may be used as a type of alternative medicine for the treatment of bone fracture healing.     

Endoglin promotes TGF-β/Smad1 signaling in scleroderma fibroblasts

TGF-β is the primary inducer of extracellular matrix proteins in scleroderma (systemic sclerosis, SSc). Previous studies indicate that in a subset of SSc fibroblasts TGF-β signaling is activated via elevated levels of activin receptor-like kinase (ALK) 1 and phosphorylated Smad1 (pSmad1). The goal of this study was to determine the role of endoglin/ALK1 in TGF-β/Smad1 signaling in SSc fibroblasts. In SSc fibroblasts, increased levels of endoglin correlated with high levels of pSmad1, collagen, and connective tissue growth factor (CCN2). Endoglin depletion via siRNA in SSc fibroblasts inhibited pSmad1 but did not affect pSmad2/3. Following endoglin depletion mRNA and protein levels of collagen and CCN2 were significantly decreased in SSc fibroblasts but remained unchanged in normal fibroblasts. ALK1 was expressed at similar levels in SSc and normal fibroblasts. Depletion of ALK1 resulted in inhibition of pSmad1 and a moderate but significant reduction of mRNA and protein levels of collagen and CCN2 in SSc fibroblasts. Furthermore, constitutively high levels of endoglin were found in complexes with ALK1 in SSc fibroblasts. Overexpression of constitutively active ALK1 (caALK1) in normal and SSc fibroblasts led to a moderate increase of collagen and CCN2. However, caALK1 potently induced endothelin 1 (ET-1) mRNA and protein levels in SSc fibroblasts. Additional experiments demonstrated that endoglin and ALK1 mediate TGF-β induction of ET-1 in SSc and normal fibroblasts. In conclusion, this study has revealed an important profibrotic role of endoglin in SSc fibroblasts. The endoglin/ALK1/Smad1 pathway could be a therapeutic target in patients with SSc if appropriately blocked.     

Canonical Wnt signaling skews TGF-β signaling in chondrocytes towards signaling via ALK1 and Smad 1/5/8

BackgroundBoth Wnt signaling and TGF-β signaling have been implicated in the regulation of the phenotype of many cell types including chondrocytes, the only cell type present in the articular cartilage. A changed chondrocyte phenotype, resulting in chondrocyte hypertrophy, is one of the main hallmarks of osteoarthritis. TGF-β signaling via activin-like kinase (ALK)5, resulting in Smad 2/3 phosphorylation, inhibits chondrocyte hypertrophy. In contrast, TGF-β signaling via ALK1, leading to Smad 1/5/8 phosphorylation, has been shown to induce chondrocyte hypertrophy. In this study, we investigated the capability of Wnt3a and WISP1, a protein downstream in canonical Wnt signaling, to skew TGF-β signaling in chondrocytes from the protective Smad 2/3 towards the Smad 1/5/8 pathway.ResultsStimulation with Wnt3a, either alone or in combination with its downstream protein WISP1, decreased TGF-β-induced C-terminal phosphorylation of Smad 2/3. In addition, both Wnt3a and WISP1 increased Smad 1/5/8 phosphorylation at the C-terminal domain in both murine and human chondrocytes. DKK-1, a selective inhibitor of canonical Wnt signaling, abolished these effects. TGF-β signaling via Smad 2/3, measured by the functional CAGA₁₂-Luc reporter construct activity, was decreased by stimulation with Wnt3a in accordance with the decrease in Smad 2/3 phosphorylation found on Western blot. Furthermore, in vivo overexpression of the canonical Wnt8a decreased Smad 2/3 phosphorylation and increased Smad 1/5/8 phosphorylation.ConclusionsOur data show that canonical Wnt signaling is able to skew TGF-β signaling towards dominant signaling via the ALK1/Smad 1/5/8 pathway, which reportedly leads to chondrocyte hypertrophy. In this way canonical Wnts and WISP1, which we found to be increased during experimental osteoarthritis, may contribute to osteoarthritis pathology.     

Protection from obesity and diabetes by blockade of TGF-β/Smad3 signaling

Imbalances in glucose and energy homeostasis are at the core of the worldwide epidemic of obesity and diabetes. Here, we illustrate an important role of the TGF-β/Smad3 signaling pathway in regulating glucose and energy homeostasis. Smad3-deficient mice are protected from diet-induced obesity and diabetes. Interestingly, the metabolic protection is accompanied by Smad3(-)(/-) white adipose tissue acquiring the bioenergetic and gene expression profile of brown fat/skeletal muscle. Smad3(-/-) adipocytes demonstrate a marked increase in mitochondrial biogenesis, with a corresponding increase in basal respiration, and Smad3 acts as a repressor of PGC-1α expression. We observe significant correlation between TGF-β1 levels and adiposity in rodents and humans. Further, systemic blockade of TGF-β signaling protects mice from obesity, diabetes, and hepatic steatosis. Together, these results demonstrate that TGF-β signaling regulates glucose tolerance and energy homeostasis and suggest that modulation of TGF-β activity might be an effective treatment strategy for obesity and diabetes.     

Overexpressed GRP78 affects EMT and cell-matrix adhesion via autocrine TGF-β/Smad2/3 signaling

Glucose-regulated protein of 78 kD (GRP78) is a multifunctional protein belonging to the heat shock protein 70 family. Overexpression of GRP78 triggered by environmental and physiological stresses is positively correlated with the occurrence and progression of various tumors, but the molecular mechanisms have not been well established. The present study indicated that overexpression of GRP78 in colon cancer cells could promote cell-matrix adhesion through the upregulation of fibronectin, integrin-β1 and phosphorylated FAK. Meanwhile, it resulted in a visible epithelial–mesenchymal transition in DLD1 cells, and the Snail-2 played the key role during the process. More importantly, the data indicated that GRP78 overexpression facilitated the expression and secretion of TGF-β1, which further activated the downstream Smad2/3 signaling module to effectuate the cell-matrix adhesion and epithelial–mesenchymal transition. Taken together, this study provides a novel molecular mechanism involving in the effects of GRP78 on colon cancer metastasis.     

Mesenchymal stem cells modified with stromal cell-derived factor 1α improve cardiac remodeling via paracrine activation of hepatocyte growth factor in a rat model of myocardial infarction

Mesenchymal stem cells (MSCs) are a promising source for cell-based treatment of myocardial infarction (MI), but existing strategies are restricted by low cell survival and engraftment. We examined whether SDF-1 transfection improve MSC viability and paracrine action in infarcted hearts. We found SDF-1-modified MSCs effectively expressed SDF-1 for at least 21days after exposure to hypoxia. The apoptosis of Ad-SDF-1-MSCs was 42% of that seen in Ad-EGFP-MSCs and 53% of untreated MSCs. In the infarcted hearts, the number of DAPI-labeling cells in the Ad-SDF-1-MSC group was 5-fold that in the Ad-EGFP-MSC group. Importantly, expression of antifibrotic factor, HGF, was detected in cultured MSCs, and HGF expression levels were higher in Ad-SDF-MSC-treated hearts, compared with Ad-EGFP-MSC or control hearts. Compared with the control group, Ad-SDF-MSC transplantation significantly decreased the expression of collagens I and III and matrix metalloproteinase 2 and 9, but heart function was improved in d-SDF-MSC-treated animals. In conclusion, SDF-1-modified MSCs enhanced the tolerance of engrafted MSCs to hypoxic injury in vitro and improved their viability in infarcted hearts, thus helping preserve the contractile function and attenuate left ventricle (LV) remodeling, and this may be at least partly mediated by enhanced paracrine signaling from MSCs via antifibrotic factors such as HGF.     

17β-Estradiol mediates TGFBR3/Smad2/3 signaling to attenuate the fibrosis of TGF-β1-induced bovine endometrial epithelial cells via GPER

Abnormal function and fibrosis of endometrium caused by cows' endometritis pose difficult implantation of embryos and uterine cavity adhesions. 17β-Estradiol (E2) serves as the most effective aromatized estrogen, and its synthetase and receptors have been detected in the endometrium. Studies have demonstrated the positive role of estrogen in combating pathological fibrosis in diverse diseases. However, it is still unknown whether E2 regulates endometrium fibrosis in bovine endometritis. Herein, we evaluated the expression patterns of transforming growth factor-β1 (TGF-β1), epithelial-mesenchymal transformation (EMT)-related proteins (α-SMA, vimentin N-cadherin and E-cadherin), cytochrome P450 19A1 (CYP19A1), and G protein-coupled estrogen receptor (GPER) in bovine healthy endometrium and Inflammatory endometrium. Our data showed that the inflamed endometrium presented low CYP19A1 and GPER expression, and significantly higher EMT process versus the normal tissue. Moreover, we established a TGF-β1-induced fibrosis model in BEND cells, and found that E2 inhibited the EMT process of BEND cells in a dose-dependent manner. The anti-fibrotic effect of E2 was blocked by the GPER inhibitor G15, but not the estrogen nuclear receptors (ERs) inhibitor ICI182780. Moreover, the GPER agonist G1 inhibited fibrosis and Smad2/3 phosphorylation but increased the expression of TGFBR3 in BEND cells. Transfection with TGFBR3 small interfering RNA blocked the effect of G1 on fibrosis of BEND cells and upregulated the expression of P-Smad2/3. Our in vivo data also showed that E2 and G1 affected uterus fibrosis in mice endometritis model caused by LPS, which was associated with the inhibition of TGFBR3/Smad2/3 signaling. In conclusion, our data implied that E2 alleviates the fibrosis of TGF-β1-induced BEND cells, which is associated with the GPER mediation of TGFBR3/Smad2/3 signaling.