Discovery of MDV6058 (PF-06952229), a selective and potent TGFßR1 inhibitor: Design, synthesis and optimization.

Compound 1 is a potent TGF-ß receptor type-1 (TGFßR1 or ALK5) inhibitor but is metabolically unstable. A solvent-exposed part of this molecule was used to analogue and modulate cell activity, liver microsome stability and mouse pharmacokinetics. The evolution of SAR that led to the selection of 2 (MDV6058 / PF-06952229) as a preclinical lead compound is described.

Trehalose attenuates TGF-ß1-induced fibrosis of hSCFs by activating autophagy.

Conjunctival fibrosis is a process of extracellular matrix accumulation and the appearance of myofibroblasts in subconjunctival fibroblasts induced by injury or inflammation, which can significantly reduce the filtration efficiency of glaucoma filtration surgery. In this study, autophagy was confirmed to be involved in regulating the fibrosis of human subconjunctival fibroblasts (hSCFs) induced by TGF-ß1. Following the addition of rapamycin, we detected that autophagy activation could reduce the increased expression level of αSMA and the accumulation of extracellular matrix component proteins namely fibronectin and type I collagen induced by TGF-ß1 via the inhibition of SMAD2 phosphorylation. Following the addition of HCQ, the inhibition of autophagy aggravated TGF-ß1-induced fibrosis of hSCFs. We further verified that trehalose, a safe clinical drug, could alleviate TGF-ß1-induced fibrosis of hSCFs by activating autophagy and that these effects could be blocked by autophagy inhibition. In summary, autophagy was shown to be involved in the regulation of TGF-ß1-induced fibrosis of hSCFs, which provided a novel perspective for exploring the progression of this lesion. More importantly, the protective effects of trehalose on TGF-ß1-induced fibrosis of hSCFs were mediated by the activation of autophagy and could provide possible new approaches for the clinical treatment of conjunctival fibrosis.

The Activation Status of the TGF-ß Transducer Smad2 Is Associated with a Reduced Survival in Gastrointestinal Cancers: A Systematic Review and Meta-Analysis.

Aberrant function of Smad2, a crucial member of transforming growth factor beta (TGF-ß) signaling, is associated with the development of malignancies, particularly in the gastrointestinal district. However, little is known about its possible prognostic role in such tumor types. With the first meta-analysis on this topic, we demonstrated that the lack of the activated form of Smad2 (phosphor-Smad2 or pSmad2), which was meant to be the C-terminally phosphorylated form, showed a statistically significant association with an increased risk of all-cause mortality in patients with gastrointestinal cancers (RR, 1.58; 95% CI, 1.05-2.37, p = 0.029, I2 = 84%), also after having adjusted for potential confounders (RR, 1.65; 95% CI, 1.24-2.18; p < 0.001; I2 = 4%). This finding highlights the importance of the TGF-ß signaling in this type of cancer. In this line, further studies are needed to explore more in depth this important molecular pathway, focusing also on potential therapeutic strategies based on its effectors or molecular targets.

Biomarker and Histopathology Evaluation of Patients with Recurrent Glioblastoma Treated with Galunisertib, Lomustine, or the Combination of Galunisertib and Lomustine.

Galunisertib, a Transforming growth factor-ßRI (TGF-ßRI) kinase inhibitor, blocks TGF-ß-mediated tumor growth in glioblastoma. In a three-arm study of galunisertib (300 mg/day) monotherapy (intermittent dosing; each cycle =14 days on/14 days off), lomustine monotherapy, and galunisertib plus lomustine therapy, baseline tumor tissue was evaluated to identify markers associated with tumor stage (e.g., histopathology, Ki67, glial fibrillary acidic protein) and TGF-ß-related signaling (e.g., pSMAD2). Other pharmacodynamic assessments included chemokine, cytokine, and T cell subsets alterations. 158 patients were randomized to galunisertib plus lomustine (n = 79), galunisertib (n = 39) and placebo+lomustine (n = 40). In 127 of these patients, tissue was adequate for central pathology review and biomarker work. Isocitrate dehydrogenase (IDH1) negative glioblastoma patients with baseline pSMAD2⁺ in cytoplasm had median overall survival (OS) 9.5 months vs. 6.9 months for patients with no tumor pSMAD2 expression (p = 0.4574). Eight patients were IDH1 R132H⁺ and had a median OS of 10.4 months compared to 6.9 months for patients with negative IDH1 R132H (p = 0.5452). IDH1 status was associated with numerically higher plasma macrophage-derived chemokine (MDC/CCL22), higher whole blood FOXP3, and reduced tumor CD3⁺ T cell counts. Compared to the baseline, treatment with galunisertib monotherapy preserved CD4⁺ T cell counts, eosinophils, lymphocytes, and the CD4/CD8 ratio. The T-regulatory cell compartment was associated with better OS with MDC/CCL22 as a prominent prognostic marker.

Associations of the Transforming Growth Factor ß/Smad Pathway, Body Mass Index, and Physical Activity With Breast Cancer Outcomes: Results From the Shanghai Breast Cancer Study.

The transforming growth factor ß (TGF-ß) pathway plays an important role in breast cancer progression and in metabolic regulation and energy homeostasis. The prognostic significance of TGF-ß interaction with obesity and physical activity in breast cancer patients remains unclear. We evaluated the expression of TGF-ß type II receptor and pSmad2 immunohistochemically in breast cancer tissue from 1,045 patients in the Shanghai Breast Cancer Study (2002-2005). We found that the presence of nuclear pSmad2 in breast cancer cells was inversely associated with overall and disease-free survival, predominantly among participants with lower body mass index (BMI; weight (kg)/height (m)2) and a moderate level of physical activity. However, the test for multiplicative interaction produced a significant result only for BMI (for disease-free survival and overall survival, adjusted hazard ratios were 1.79 and 2.05, respectively). In 535 earlier-stage (T1-2, N0) invasive cancers, nuclear pSmad2 was associated with improved survival among persons with higher BMI (overall survival: adjusted hazard ratio = 0.27, 95% confidence interval: 0.09, 0.86). The cytoplasmic pattern of TGF-ß type II receptor expression in cancer cells was significantly associated with a lower survival rate but was not modified by BMI or physical activity. Our study suggests that the TGF-ß pathway in tumor cells is involved in breast cancer prognosis and may be modified by BMI through pSmad2.

Axin1 and Axin2 regulate the WNT-signaling landscape to promote distinct mesoderm programs.

How distinct mesodermal lineages - extraembryonic, lateral, intermediate, paraxial and axial - are specified from pluripotent epiblast during gastrulation is a longstanding open question. By investigating AXIN, a negative regulator of the WNT/ß-catenin pathway, we have uncovered new roles for WNT signaling in the determination of mesodermal fates. We undertook complementary approaches to dissect the role of WNT signaling that augmented a detailed analysis of Axin1;Axin2 mutant mouse embryos, including single-cell and single-embryo transcriptomics, with in vitro pluripotent Epiblast-Like Cell differentiation assays. This strategy allowed us to reveal two layers of regulation. First, WNT initiates differentiation of primitive streak cells into mesoderm progenitors, and thereafter, WNT amplifies and cooperates with BMP/pSMAD1/5/9 or NODAL/pSMAD2/3 to propel differentiating mesoderm progenitors into either posterior streak derivatives or anterior streak derivatives, respectively. We propose that Axin1 and Axin2 prevent aberrant differentiation of pluripotent epiblast cells into mesoderm by spatially and temporally regulating WNT signaling levels.

Smad7 foci are present in micronuclei induced by heavy particle radiation.

DNA damage and reactive oxygen species (ROS) generated by ionizing radiation (IR) activate DNA damage response (DDR) and cytokine signaling pathways, including double strand break (DSB) repair and TGFß/Smad signaling pathway. Proteins assembled at IR-induced DSB sites can be visualized as foci, including γH2AX, 53BP1, ATM and ATF2. Unrepaired DSBs are thought to be one origin of micronuclei (MN), an indicator of genotoxic stress and chromosomal instability. Studies have detected γH2AX in IR-induced MN, indicating the presence of DSB in MN. Previously we reported that TGFß downstream proteins Smad7 and phospho-Smad2 (pSmad2) co-localized with DDR proteins following radiation. Here we studied the status of Smad7 and pSmad2 in MN post high linear energy transfer (LET) radiation in human normal and cancerous cells. We observed γH2AX foci in IR-induced MN, whereas 53BP1 and ATF2 were absent. Interestingly, Smad7 foci, but not pSmad2, were detectable in both spontaneous and IR-induced MN. We compared the effect of particle track structures on the yield of MN using 5.6MeV/u boron (B) and 600MeV/u iron (Fe) particles with similar LET (200 and 180keV/µm, respectively) in human fibroblasts. The frequency of MN induced by B was lower than that by Fe particles, albeit the proportion of Smad7-positive to Smad7-negative MN remained constant. An increased frequency of spontaneous MN, with slightly higher ratio of Smad7 or γH2AX positive, was found in human prostate cancer cells (PC3) compared to normal cells. 24h after 1Gy of Fe particles exposure, the yield of MN increased, and the majority (∼70%) carried γH2AX and Smad7. Phospho-ATM (Ser1981) foci were found in both spontaneous and IR-induced MN in PC3 cells, displaying a much lower frequency compared to γH2AX and Smad7. Our data suggest a unique role of Smad7 in IR-induced MN formation, which may associate with DNA repair, apoptosis and genomic instability.

Aloin alleviates pathological cardiac hypertrophy via modulation of the oxidative and fibrotic response.

AIMS: Pathological cardiac hypertrophy is a characteristic feature in many cardiovascular diseases (CVDs). Aloin is an anthraquinone glycoside from Aloe species, and the effect of aloin on cardiac hypertrophy and associated fibrotic changes have not been elucidated. This study investigated the effect of aloin against the isoproterenol (ISO)-induced cardiac hypertrophy in rats. MAIN METHODS: Cardiac hypertrophy experimental model was induced in rats by subcutaneous injection of ISO for 14 days. Meanwhile, the animals were administered orally with aloin at doses of 25 and 50 mg/kg/day. On the 15th day, cardiac echocardiography was performed, the heart was collected and subjected for histopathological, gene expression, and immunoblot studies. Additionally, the effect of aloin on ISO-induced hypertrophic changes in H9c2 cells was investigated. KEY FINDINGS: Aloin markedly alleviated ISO-induced heart injury, reduced cardiac hypertrophy, improved cardiac function, and histological alterations in the heart. Mechanistically, aloin attenuated ISO-induced fibrosis via inhibition of the levels of collagen I, α-smooth muscle actin (α-SMA), fibronectin, transforming growth factor-ß (TGF-ß) and pSmad2/3 proteins in the heart. Aloin alleviated ISO-induced myocardial oxidative damage and up-regulated the levels of antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) proteins. Moreover, aloin treatment attenuated ISO-induced hypertrophic changes and the generation of reactive oxygen species (ROS) in H9c2 cells in vitro. SIGNIFICANCE: Our findings demonstrated that aloin alleviated ISO-induced cardiac hypertrophy and fibrosis via inhibiting TGF-ß/pSmad2/3 signaling and restoring myocardial antioxidants, and therefore has promising therapeutic potential against cardiac hypertrophy and fibrosis.

Reduced Proteolytic Cleavage of von Willebrand Factor Leads to Aortic Valve Stenosis and Load-Dependent Ventricular Remodeling.

We hypothesized that excess endothelial-associated von Willebrand factor (vWF) and secondary platelet adhesion contribute to aortic valve stenosis (AS). We studied hyperlipidemic mice lacking ADAMTS13 (LDLR -/- AD13 -/- ), which cleaves endothelial-associated vWF multimers. On echocardiography and molecular imaging, LDLR -/- AD13 -/- compared with control strains had increased aortic endothelial vWF and platelet adhesion and developed hemodynamically significant AS, arterial stiffening, high valvulo-aortic impedance, and secondary load-dependent reduction in LV systolic function. Histology revealed leaflet thickening and calcification with valve interstitial cell myofibroblastic and osteogenic transformation, and evidence for TGFß1 pathway activation. We conclude that valve leaflet endothelial vWF-platelet interactions promote AS through juxtacrine platelet signaling.

Allergic asthma aggravates angiotensin â ¡-induced cardiac remodeling in mice.

Cardiovascular disease remains the leading cause of death globally, and heart failure (HF) represents its terminal stage. Asthma, one of the most common chronic diseases, has been reported to be associated with an increased risk of cardiovascular disease. However, the link between asthma and HF has rarely been studied, and the possible mechanisms by which asthma affects HF are unclear. This study aimed to explore the influence of asthma on HF and the possible mechanisms. We analyzed data from the National Health and Nutrition Examination Survey and found a higher prevalence of HF among asthmatic individuals, and identified an independent association between HF and asthma. Subsequently, we produced mice with concurrent ovalbumin (OVA) sensitization-induced allergic asthma and angiotensin Ⅱ infusion-induced cardiac remodeling to explore the effect of asthma on cardiac remodeling in vivo. The results showed that OVA-induced asthma impaired heart function and aggravated cardiac remodeling in mice. We also found that OVA sensitization increased the expression levels of immunoglobulin E (IgE) in serum and IgE receptor (FcεR1) in the heart, and enhanced the activation of downstream signaling molecules of IgE-FcεR1 in the heart. Importantly, blockage of IgE-FcεR1 using FcεR1-deficient mice or an anti-IgE antibody prevented asthma-induced decline of cardiac function, and alleviated cardiac remodeling. These findings demonstrate the adverse effects of allergic asthma on the heart, and suggest the potential application of anti-IgE therapy in the treatment of asthma complicated with heart conditions.

Effects of oral losartan administration on homeostasis of articular cartilage and bone in a rabbit model.

Background and aims: Previous work has shown that oral losartan can enhance microfracture-mediated cartilage repair in a rabbit osteochondral defect injury model. In this study, we aimed to determine whether oral losartan would have a detrimental effect on articular cartilage and bone homeostasis in the uninjured sides. Methods: New Zealand rabbits were divided into 4 groups including normal uninjured (Normal), contralateral uninjured side of osteochondral defect (Defect), osteochondral defect plus microfracture (Microfracture) and osteochondral defect plus microfracture and losartan oral administration (10 mg/kg/day) (Losartan). Rabbits underwent different surgeries and treatment and were sacrificed at 12 weeks. Both side of the normal group and uninjured side of treatment groups tibias were harvested for Micro-CT and histological analysis for cartilage and bone including H&E staining, Herovici's staining (bone and cartilage) Alcian blue and Safranin O staining (cartilage) as well as immunohistochemistry of losartan related signaling pathways molecules for both cartilage and bone. Results: Our results showed losartan oral treatment at 10 mg/kg/day slightly increase Alcian blue positive matrix as well as decrease collagen type 3 in articular cartilage while having no significant effect on articular cartilage structure, cellularity, and other matrix. Losartan treatment also did not affect angiotensin receptor type 1 (AGTR1), angiotensin receptor type 2 (AGTR2) and phosphorylated transforming factor ß1 activated kinase 1 (pTAK1) expression level and pattern in the articular cartilage. Furthermore, losartan treatment did not affect microarchitecture of normal cancellous bone and cortical bone of tibias compared to normal and other groups. Losartan treatment slightly increased osteocalcin positive osteoblasts on the surface of cancellous bone and did not affect bone matrix collagen type 1 content and did not change AGTR1, AGTR2 and pTAK1 signal molecule expression. Conclusion: Oral losartan used as a microfracture augmentation therapeutic does not have significant effect on uninjured articular cartilage and bone based on our preclinical rabbit model. These results provided further evidence that the current regimen of using losartan as a microfracture augmentation therapeutic is safe with respect to bone and cartilage homeostasis and support clinical trials for its application in human cartilage repair.

Smad3 gene C-terminal phosphorylation site mutation exacerbates CCl4-induced hepatic fibrogenesis by promoting pSmad2L/C-mediated signaling transduction.

Current researches have confirmed that Smads, mediators of TGF-ß signaling, are strictly controlled by domain-specific site phosphorylation in the process of hepatic disease. Usually, Smad3 phospho-isoform pSmad3L and pSmad3C are reversible and antagonistic; pSmad2L/C could act together with pSmad3L by stimulating PAI-1 expression and ECM synthesis to transmit fibrogenic signals. Our recent study found that pSmad3C mutation is supposed to perform a vigorous role on the early phase of liver injury and abates salvianolic acid B's anti-hepatic fibrotic-carcinogenesis. However, whether pSmad3C mutation expedites pSmad2L/C-mediated signaling transduction during hepatic fibrogenesis remains vague. Presently, Smad3 gene C-terminal phosphorylation site mutation heterozygote (pSmad3C+/-) mice were constructed to probe if and how pSmad3C retards CCl4-induced hepatic fibrogenesis by inhibiting pSmad2L/C-mediated signaling transduction. Twelve 6-week-old pSmad3C+/- C57BL/6J mice were intraperitoneally injection with CCl4 for 6 weeks to induce liver fibrogenesis. Results showed that pSmad3C mutation aggravates the relative liver weight, biochemical parameters, collagenous fibers and fibrotic septa formation, contributes to fibrogenesis in HT-CCl4 mice. Furthermore, fibrotic-related proteins TGF-ß1, pSmad2C, pSmad2L, and PAI-1 were also increased in CCl4-induced pSmad3C+/- mice. These results suggest that pSmad3C mutation exacerbates hepatic fibrogenesis which relates to intensifying pSmad2L/C-mediated signaling transduction.

Activation of Nrf2/AREs-mediated antioxidant signalling, and suppression of profibrotic TGF-ß1/Smad3 pathway: a promising therapeutic strategy for hepatic fibrosis - A review.

Hepatic fibrosis (HF) is a wound-healing response that occurs during chronic liver injury and features by an excessive accumulation of extracellular matrix (ECM) components. Activation of hepatic stellate cell (HSC), the leading effector in HF, is responsible for overproduction of ECM. It has been documented that transforming growth factor-ß1 (TGF-ß1) stimulates superfluous accumulation of ECM and triggers HSCs activation mainly via canonical Smad-dependent pathway. Also, the pro-fibrogenic TGF-ß1 is correlated with generation of reactive oxygen species (ROS) and inhibition of antioxidant mechanisms. Moreover, involvement of oxidative stress (OS) can be clearly elucidated as a fundamental event in liver fibrogenesis. Nuclear factor erythroid 2-related factor 2-antioxidant response elements (Nrf2-AREs) pathway, a group of OS-mediated transcription factors with diverse downstream targets, is associated with the induction of diverse detoxifying enzymes and the most pivotal endogenous antioxidative system. More specifically, Nrf2-AREs pathway has recently assigned as a new therapeutic target for cure of HF. The overall goal of this review will focus on recent findings about activation of Nrf2-AREs-mediated antioxidant and suppression of profibrotic TGF-ß1/Smad3 pathway in the liver, providing an overview of recent advances in transcriptional repressors that dislocated during HF formation, and highlighting possible novel therapeutic targets for liver fibrosis.

The Relationship between Nkx2.1 and DNA Oxidative Damage Repair in Nickel Smelting Workers: Jinchang Cohort Study.

Background: Occupational nickel exposure can cause DNA oxidative damage and influence DNA repair. However, the underlying mechanism of nickel-induced high-risk of lung cancer has not been fully understood. Our study aims to evaluate whether the nickel-induced oxidative damage and DNA repair were correlated with the alterations in Smad2 phosphorylation status and Nkx2.1 expression levels, which has been considered as the lung cancer initiation gene. Methods: 140 nickel smelters and 140 age-matched administrative officers were randomly stratified by service length from Jinchang Cohort. Canonical regression, χ² test, Spearman correlation etc. were used to evaluate the association among service length, MDA, 8-OHdG, hOGG1, PARP, pSmad2, and Nkx2.1. Results: The concentrations of MDA, PARP, pSmad2, and Nkx2.1 significantly increased. Nkx2.1 (rs = 0.312, p < 0.001) and Smad2 phosphorylation levels (rs = 0.232, p = 0.006) were positively correlated with the employment length in nickel smelters, which was not observed in the administrative officer group. Also, elevation of Nkx2.1 expression was positively correlated with service length, 8-OHdG, PARP, hOGG1 and pSmad2 levels in nickel smelters. Conclusions: Occupational nickel exposure could increase the expression of Nkx2.1 and pSmad2, which correlated with the nickel-induced oxidative damage and DNA repair change.

Tumor necrosis factor-related apoptosis-inducing ligand regulate the accumulation of extracelluar matrix in pulmonary artery by activating the phosphorylation of Smad2/3.

Introduction: Previous studies have found that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was involved in the progression of pulmonary hypertension (PH), and TRAIL knocking (KO) has an inhibitory effect on PH, but its mechanism is not completely clear. Methods: The effects of TRAIL on the accumulation of extracelluar matrix (ECM), which is one of the most important processes of vascular remodeling, were observed in mice and isolated pulmonary artery smooth muscle cells (PASMCs). In vivo, mice were divided into four groups: Control group (n = 5), hypoxia-induced PH mice group (n = 8), anti-TRAIL antibody (TRAIL-Ab) treatment group (n = 8) and IgG antibody (IgG) group (n = 8). The effects of TRAIL-Ab on ECM expression in hypoxic induced PH were researched; in vivo, PASMCs were divided into three groups: Control group, hypoxia-induced group, TRAIL-Ab group. Expressions of p-Smad2/3 and p-Smad1/5/8 were compared among the three groups. Results: Hypoxia-induced PH mice had significant increases in right ventricle systolic pressure (RVSP) (P < 0.001), right ventricular hypertrophy (RVH) (P = 0.007), vascular stenosis (P < 0.001) compared with controls. Mice with anti-TRAIL antibody had lower levels in RVSP (P < 0.001), RVH (P < 0.001), vascular stenosis (P < 0.001) than PH mice. Besides, the TRAIL-Ab significantly inhibited the phosphorylation of Smad2/3 compared with hypoxia-induced group. Conclusion: TRAIL regulates the accumulation of ECM in pulmonary artery by activating pSmad2/3.

The Interaction of CDH20 With ß-Catenin Inhibits Cervical Cancer Cell Migration and Invasion via TGF-ß/Smad/SNAIL Mediated EMT.

Cancer-associated cadherin 20 (CDH20) is a novel identified cadherin that is genetically altered in several types of human cancer, including cervical cancer. However, its involvement in the progression of cervical cancer remains unknown. In this study, we show that CDH20 was downregulated in clinical cervical cancer samples and its expression correlated with cervical cancer clinical features. CDH20 negatively regulated the migration and invasion of cervical cancer cells. CDH20 increased the expression and promoted the cytoplasm and membrane translocation of ß-catenin, and interacted with ß-catenin. Mechanistically, CDH20/ß-catenin suppressed transforming growth factor-ß (TGF-ß)-induced epithelial-to-mesenchymal transition (EMT) by downregulating Snail through reducing the phosphorylation and nuclear translocation of Smad2/3. Taken together, our data suggest that CDH20 may act as a tumor suppressor that interacts with ß-catenin to inhibit cervical cancer cell migration and invasion via TGF-ß/Smad/Snail mediated EMT.

miRNA-23a/27a attenuates muscle atrophy and renal fibrosis through muscle-kidney crosstalk.

BACKGROUND: The treatment of muscle wasting is accompanied by benefits in other organs, possibly resulting from muscle-organ crosstalk. However, how the muscle communicates with these organs is less understood. Two microRNAs (miRs), miR-23a and miR-27a, are located together in a gene cluster and regulate proteins that are involved in the atrophy process. MiR-23a/27a has been shown to reduce muscle wasting and act as an anti-fibrotic agent. We hypothesized that intramuscular injection of miR-23a/27a would counteract both muscle wasting and renal fibrosis lesions in a streptozotocin-induced diabetic model. METHODS: We generated an adeno-associated virus (AAV) that overexpresses the miR-23a∼27a∼24-2 precursor RNA and injected it into the tibialis anterior muscle of streptozotocin-induced diabetic mice. Muscle cross-section area (immunohistology plus software measurement) and muscle function (grip strength) were used to evaluate muscle atrophy. Fibrosis-related proteins were measured by western blot to monitor renal damage. In some cases, AAV-GFP was used to mimic the miR movement in vivo, allowing us to track organ redistribution by using the Xtreme Imaging System. RESULTS: The injection of AAV-miR-23a/27a increased the levels of miR-23a and miR-27a as well as increased phosphorylated Akt, attenuated the levels of FoxO1 and PTEN proteins, and reduced the abundance of TRIM63/MuRF1 and FBXO32/atrogin-1 in skeletal muscles. It also decreased myostatin mRNA and protein levels as well as the levels of phosphorylated pSMAD2/3. Provision of miR-23a/27a attenuates the diabetes-induced reduction of muscle cross-sectional area and muscle function. Curiously, the serum BUN of diabetic animals was reduced in mice undergoing the miR-23a/27a intervention. Renal fibrosis, evaluated by Masson trichromatic staining, was also decreased as were kidney levels of phosphorylated SMAD2/3, alpha smooth muscle actin, fibronectin, and collagen. In diabetic mice injected intramuscularly with AAV-GFP, GFP fluorescence levels in the kidneys showed linear correlation with the levels in injected muscle when examined by linear regression. Following intramuscular injection of AAV-miR-23a∼27a∼24-2, the levels of miR-23a and miR-27a in serum exosomes and kidney were significantly increased compared with samples from control virus-injected mice; however, no viral DNA was detected in the kidney. CONCLUSIONS: We conclude that overexpression of miR-23a/27a in muscle prevents diabetes-induced muscle cachexia and attenuates renal fibrosis lesions via muscle-kidney crosstalk. Further, this crosstalk involves movement of miR potentially through muscle originated exosomes and serum distribution without movement of AAV. These results could provide new approaches for developing therapeutic strategies for diabetic nephropathy with muscle wasting.

Significance of pSmad2/3 and Smad4 in hepatitis C virus-related liver fibrosis and hepatocellular carcinoma.

Chronic hepatitis C (CHC) is a major public health problem, especially in Egypt. Risk of hepatocellular carcinoma (HCC) development increases as hepatitis C virus (HCV)-related liver diseases progress. Smads act as substrates for the transforming growth factor-beta (TGF-ß) family of receptors. This study aims to assess hepatic expression of pSmad2/3 and Smad4 in CHC with different stages of fibrosis and grades of necro-inflammation as well as in HCC on top of CHC. This study was done on 33 core liver biopsies from patients with CHC (15 with early fibrosis and 18 with late fibrosis), 15 liver specimens from HCC cases on top of CHC, as well as five normal controls. pSmad2/3 and Smad4 show more immunopositivity, higher percentage of positive hepatocytes and stronger staining intensity in CHC with late fibrosis compared to early fibrosis. pSmad2/3 shows increase of the previous parameters in CHC with high grade activity than those with low activity. Smad4 shows increase of the previous parameters in HCC compared to CHC cases. pSmad2/3 and Smad4 can be used as diagnostic and/or prognostic markers for progression of HCV-related fibrosis to cirrhosis and further progression to HCC.

Tumor-promoting role of Porphyromonas gingivalis fimbrillin in progression of esophageal squamous cell cancer / 中华微生物学和免疫学杂志

Objective:To investigate the effects of Porphyromonas gingivalis ( P. gingivalis) fimbrillin (FimA) on the progression of esophageal squamous cell carcinoma (ESCC). Methods:Wild-type P. gingivalis and fimA gene-deleted P. gingivalis ( fimA-/-P. gingivalis) were used to infect ESCC cells after morphology and PCR identification. Immunofluorescence, CCK-8 and Transwell chamber were used to detect the effects of FimA on the infectivity of P. gingivalis and it influences on cell invasion, proliferation and migration. Western blot was used to detect pSmad2/3 changes. The growth of tumor was detected in a nude mouse model bearing subcutaneous tumor. Results:Deletion of FimA might reduce the interbacterial adhesion of P. gingivalis. Compared with wild-type P. gingivalis, less fimA-/-P. gingivalis could infect NE6-T cells. Moreover, the proliferation, migration and invasion of NE6-T and KYSE30 cells as well as the activation of pSmad2/3 induced by P. gingivalis were inhibited after deletion of FimA. The growth of KYSE30 infected by fimA-/-P. gingivalis in nude mice was significantly slower than that of the wild-type P. gingivalis group. Conclusions:FimA mediated the effects of P. gingivalis on promoting the evolution of ESCC and was a potential target molecule to block the tumor-promoting effect of P. gingivalis.

Transforming growth factor-ß promotes aggressiveness and invasion of clear cell renal cell carcinoma.

The molecular mechanisms whereby transforming growth factor-ß (TGF-ß) promotes clear cell renal cell carcinoma (ccRCC) progression is elusive. The cell membrane bound TGF-ß type I receptor (ALK5), was recently found to undergo proteolytic cleavage in aggressive prostate cancer cells, resulting in liberation and subsequent nuclear translocation of its intracellular domain (ICD), suggesting that ALK5-ICD might be a useful cancer biomarker. Herein, the possible correlation between ALK5 full length (ALK5-FL) and ALK5-ICD protein, phosphorylated Smad2/3 (pSmad2/3), and expression of TGF-ß target gene PAI-1, was investigated in a clinical ccRCC material, in relation to tumor grade, stage, size and cancer specific survival. Expression of ALK5-FL, ALK5-ICD, pSmad2/3 and PAI-1 protein levels were significantly higher in higher stage and associated with adverse survival. ALK5-ICD, pSmad2/3 and PAI-1 correlated with higher grade, and ALK5-FL, pSmad2/3 and PAI-1 protein levels were significantly correlated with larger tumor size. Moreover, the functional role of the TGF-ß - ALK5-ICD pathway were investigated in two ccRCC cell lines by treatment with ADAM/MMP2 inhibitor TAPI-2, which prevented TGF-ß-induced ALK5-ICD generation, nuclear translocation, as well as cell invasion. The present study demonstrated that canonical TGF-ß Smad2/3 pathway and generation of ALK5-ICD correlates with poor survival and invasion of ccRCC in vitro.