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Clopidogrel, a P2Y12 inhibitor, is a novel anti-fibrosis agent for chronic kidney disease (CKD), but its mechanisms remain unclear, which we investigated by silencing P2Y12 or treating unilateral ureteral obstruction (UUO) in LysM-Cre/Rosa Tomato mice with clopidogrel in vivo and in vitro. We found that P2Y12 was significantly increased and correlated with progressive renal fibrosis in CKD patients and UUO mice. Phenotypically, up to 82% of P2Y12-expressing cells within the fibrosing kidney were of macrophage origin, identified by co-expressing CD68/F4/80 antigens or a macrophage-lineage-tracing marker Tomato. Unexpectedly, more than 90% of P2Y12-expressing macrophages were undergoing macrophage-to-myofibroblast transition (MMT) by co-expressing alpha smooth muscle actin (α-SMA), which was also confirmed by single-cell RNA sequencing. Functionally, clopidogrel improved the decline rate of the estimated glomerular filtration rate (eGFR) in patients with CKD and significantly inhibited renal fibrosis in UUO mice. Mechanistically, P2Y12 expression was induced by transforming growth factor β1 (TGF-β1) and promoted MMT via the Smad3-dependent mechanism. Thus, silencing or pharmacological inhibition of P2Y12 was capable of inhibiting TGF-β/Smad3-mediated MMT and progressive renal fibrosis in vivo and in vitro. In conclusion, P2Y12 is highly expressed by macrophages in fibrosing kidneys and mediates renal fibrosis by promoting MMT via TGF-β/Smad3 signaling. Thus, P2Y12 inhibitor maybe a novel and effective anti-fibrosis agent for CKD.

Tumor-associated macrophages (TAM) have important roles in cancer progression, but the signaling behind the formation of protumoral TAM remains understudied. Here, by single-cell RNA sequencing, we revealed that the pattern recognition receptor Mincle was highly expressed in TAM and significantly associated with mortality in patients with non–small cell lung cancer. Cancer cells markedly induced Mincle expression in bone marrow–derived macrophages (BMDM), thus promoting cancer progression in invasive lung carcinoma LLC and melanoma B16F10 in vivo and in vitro. Mincle was predominately expressed in the M2-like TAM in non–small cell lung carcinoma and LLC tumors, and silencing of Mincle unexpectedly promoted M1-like phenotypes in vitro. Mechanistically, we discovered a novel Mincle/Syk/NF-κB signaling pathway in TAM needed for executing their TLR4-independent protumoral activities. Adoptive transfer of Mincle-silenced BMDM significantly suppressed TAM-driven cancer progression in the LLC-bearing NOD/SCID mice. By modifying our well-established ultrasound microbubble–mediated gene transfer protocol, we demonstrated that tumor-specific silencing of Mincle effectively blocked Mincle/Syk/NF-κB signaling, therefore inhibiting the TAM-driven cancer progression in the syngeneic mouse cancer models. Thus, our findings highlight the function of Mincle as a novel immunotherapeutic target for cancer via blocking the Mincle/Syk/NF-κB circuit in TAM.

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Natural killer (NK) cells, early effectors in anticancer immunity, are paralyzed by TGFβ1, an immunosuppressive cytokine produced by cancer cells. Development and activity of NK cells are largely inhibited in the Smad3-dependent tumor microenvironment. Here, we used genetic engineering to generate a stable SMAD3-silencing human NK cell line, NK-92-S3KD, whose cancer-killing activity and cytokine production were significantly enhanced under TGFβ1-rich condition compared with the parental cell line. Interestingly, we identified that the IFNG gene is a direct E4BP4 target gene. Thus, silencing of SMAD3 allows upregulation of E4BP4 that subsequently promoting interferon-γ (IFNγ) production in the NK-92-S3KD cells. More importantly, NK-92-S3KD immunotherapy increases the production of not only IFNγ, but also granzyme B and perforin in tumors; therefore, inhibiting cancer progression in two xenograft mouse models with human hepatoma (HepG2) and melanoma (A375). Thus, the NK-92-S3KD cell line may be useful for the clinical immunotherapy of cancer. Cancer Immunol Res; 6(8); 965–77. ©2018 AACR.

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Rationale: Poor β cell proliferation is one of the detrimental factors hindering islet cell replacement therapy for patients with diabetes. Smad3 is an important transcriptional factor of TGF-β signaling and has been shown to promote diabetes by inhibiting β cell proliferation. Therefore, we hypothesize that Smad3-deficient islets may be a novel cell replacement therapy for diabetes. Methods: We examined this hypothesis in streptozocin-induced type-1 diabetic mice and type-2 diabetic db/db mice by transplanting Smad3 knockout (KO) and wild type (WT) islets under the renal capsule, respectively. The effects of Smad3KO versus WT islet replacement therapy on diabetes and diabetic kidney injury were examined. In addition, RNA-seq was applied to identify the downstream target gene underlying Smad3-regulated β cell proliferation in Smad3KO-db/db versus Smad3WT-db/db mouse islets. Results: Compared to Smad3WT islet therapy, treatment with Smad3KO islets produced a much better therapeutic effect on both type-1 and type-2 diabetes by significantly lowering serum levels of blood glucose and HbA1c and protected against diabetic kidney injuries by preventing an increase in serum creatinine and the development of proteinuria, mesangial matrix expansion, and fibrosis. These were associated with a significant increase in grafted β cell proliferation and blood insulin levels, resulting in improved glucose intolerance. Mechanistically, RNA-seq revealed that compared with Smad3WT-db/db mouse islets, deletion of Smad3 from db/db mouse islets markedly upregulated E2F3, a pivotal regulator of cell cycle G1/S entry. Further studies found that Smad3 could bind to the promoter of E2F3, and thus inhibit β cell proliferation via an E2F3-dependent mechanism as silencing E2F3 abrogated the proliferative effect on Smad3KO β cells. Conclusion: Smad3-deficient islet replacement therapy can significantly improve both type-1 and type-2 diabetes and protect against diabetic kidney injury, which is mediated by a novel mechanism of E2F3-dependent β cell proliferation.

Uterine cervical cancer, the second most frequently occurring cancer in women worldwide, is tightly associated with the expression of high-risk human papillomavirus [mainly human papillomavirus (HPV)-16 and HPV18] oncogenes E6 and E7 and characteristically exhibits chromosomal instability. However, the mechanisms underlying chromosomal instability in cervical cancer are still not fully understood. In this study, we observed that two of three human cervical epithelial cell lines expressing HPV16 E6E7 became immortalized without extensive chromosomal instability and crisis. The introduction of transforming growth factor (TGF)-β1, a multiple functional cytokine/growth factor, in the culture medium induced crisis, which was associated with massive chromosomal end-to-end fusions and other structural aberrations. The distributions of structural aberrations on individual chromosomes were significantly correlated with the profiles of telomere signal–free ends. The immortalized cells that emerged from the TGF-β1–induced crisis showed multiple clonal structural aberrations that were not observed in cells without TGF-β1 treatment. Overexpression of the catalytic subunit of telomerase (hTERT) abolished the effects of TGF-β1 on chromosomal instability. Interestingly, another HPV16 E6E7–expressing cervical cell line that experienced crisis and telomere dysfunction under ordinary culture condition had a higher level of autocrine TGF-β1 production than the other two crisis-free immortalized cell lines. Blocking the TGF-β1 pathway by an inhibitor of TGF-β1 receptor type I prevented the crisis and telomere-mediated chromosomal instability. In addition, more dramatic telomere shortening was observed in cervical intraepithelial neoplasias having higher expression of TGF-β1 in vivo. These results together suggest an important role of TGF-β1 in the early process of cervical carcinogenesis. [Cancer Res 2008;68(17):7200–9]

Supplementary Figure 3 from Transforming Growth Factor β1 Promotes Chromosomal Instability in Human Papillomavirus 16 E6E7–Infected Cervical Epithelial Cells

Supplementary Figure 4 from Transforming Growth Factor β1 Promotes Chromosomal Instability in Human Papillomavirus 16 E6E7–Infected Cervical Epithelial Cells

Supplementary Figure 1 from Transforming Growth Factor β1 Promotes Chromosomal Instability in Human Papillomavirus 16 E6E7–Infected Cervical Epithelial Cells

Supplementary Table 1 from Transforming Growth Factor β1 Promotes Chromosomal Instability in Human Papillomavirus 16 E6E7–Infected Cervical Epithelial Cells

Supplementary Figure 2 from Transforming Growth Factor β1 Promotes Chromosomal Instability in Human Papillomavirus 16 E6E7–Infected Cervical Epithelial Cells

Increasing evidence shows that SARS-CoV-2 can infect kidneys and cause acute kidney injury (AKI) in critically ill COVID-19 patients. However, mechanisms through which COVID-19 induces AKI are largely unknown and treatment remains no-effective. Here, we report that kidney-specifically overexpressing SARS-CoV-2 N gene can cause AKI including tubular necrosis and elevated levels of serum creatinine and BUN in 8-week-old diabetic db/db mice, which become worse in those with older age (16 weeks) and underlying diabetic kidney disease (DKD).Treatment with quercetin, a purified product from the traditional Chinese medicine (TCM) that shows effective treatment of COVID-19 patients, can significantly inhibit SARS-CoV-2 N protein-induced AKI in diabetic mice with or without underlying DKD. Mechanistically, quercetin can block the binding of SARS-CoV-2 N protein to Smad3, thereby inhibiting Smad3 signaling and Smad3-mediated cell death via the p16-dependent G1 cell cycle arrest mechanism in vivo and vitro. In conclusion, SARS-CoV-2 N protein is pathogenic and can cause severe AKI in diabetic mice, particularly in those with older age and pre-existing DKD, via the Smad3-dependent G1 cell cycle arrest mechanism. Importantly, we identify that quercetin may be an effective TCM compound capable of inhibiting COVID-19 AKI by blocking SARS-CoV-2 N-Smad3-mediated cell death pathway.

Tumor innervation is a common phenomenon with unknown mechanism. Here, we discovered a direct mechanism of tumor-associated macrophage (TAM) for promoting de novo neurogenesis via a subset showing neuronal phenotypes and pain receptor expression associated with cancer-driven nocifensive behaviors. This subset is rich in lung adenocarcinoma associated with poorer prognosis. By elucidating the transcriptome dynamics of TAM with single-cell resolution, we discovered a phenomenon “macrophage to neuron-like cell transition” (MNT) for directly promoting tumoral neurogenesis, evidenced by macrophage depletion and fate-mapping study in lung carcinoma models. Encouragingly, we detected neuronal phenotypes and activities of the bone marrow–derived MNT cells (MNTs) in vitro. Adoptive transfer of MNTs into NOD/SCID mice markedly enhanced their cancer-associated nocifensive behaviors. We identified macrophage-specific Smad3 as a pivotal regulator for promoting MNT at the genomic level; its disruption effectively blocked the tumor innervation and cancer-dependent nocifensive behaviors in vivo. Thus, MNT may represent a precision therapeutic target for cancer pain.

Introduction and Aims: Elevated plasma levels of C-reactive protein (CRP) are closely associated with progressive renal injury in patients with chronic kidney disease (CKD). Here, we tested a hypothesis that CRP may promote renal fibrosis and inflammation via a TGF-β/Smad3-dependent mechanism. Methods: Role and mechanisms of TGF-β/Smad3 in CRP-induced renal fibrosis and inflammation were examined in a mouse model of unilateral ureteral obstruction (UUO) induced in CRP Tg/Smad3 KO mice and in a rat tubular epithelial cell line in which Smad3 gene is stably knocked down (S3KD-NRK52E). Results: We found that mice overexpressing the human CRP gene were largely promoted renal inflammation and fibrosis as evidenced by increasing IL-1β, TNF-α, MCP-1 expression, F4/80+ macrophages infiltration, and marked accumulation of α-smooth muscle actin (α-SMA), collagen I and fibronectin in the UUO kidney, which were blunted when Smad3 gene was deleted in CRPtg-Smad3KO. Mechanistically, we found that the protection of renal inflammation and fibrosis in the UUO kidney of CRPtg-Smad3KO mice was associated with the inactivation of CD32-NF-κB and TGF-β/Smad3 signaling. Conclusion: In conclusion, Smad3 deficiency protects against CRP-mediated renal inflammation and fibrosis in the UUO kidney by inactivating CD32-NF-κB and TGF-β/Smad3 signaling.

Transforming growth factor β (TGF-β) has been shown to promote tumor invasion and metastasis by activating the matrix metalloproteinases (MMPs); however, signaling mechanisms remain controversial and therapies targeting MMPs are still suboptimal. In the present study, we found that combined therapy with Asiatic acid (AA), a Smad7 agonist, and Naringenin (NG), a Smad3 inhibitor, effectively retrieved the balance between Smad3 and Smad7 signaling in the TGF-β-rich tumor microenvironment and thus significantly suppressed tumor invasion and metastasis in mouse models of melanoma and lung carcinoma. Mechanistically, we unraveled that Smad3 acted as a transcriptional activator of MMP2 and as a transcriptional suppressor of tissue inhibitors of metalloproteinase-2 (TIMP2) via binding to 5′ UTR of MMP2 and 3′ UTR of TIMP2, respectively. Treatment with NG inhibited Smad3-mediated MMP2 transcription while increasing TIMP, whereas treatment with AA enhanced Smad7 to suppress TGF-β/Smad3 signaling, as well as the activation of MMP2 by targeting the nuclear factor-κB (NF-κB)-membrane-type-1 MMP (MT1-MMP) axis. Therefore, the combination of AA and NG additively suppressed invasion and metastasis of melanoma and lung carcinoma by targeting TGF-β/Smad-dependent MMP2 transcription, post-translational activation, and function., Graphical Abstract, TGF-β-induced MMPs are key mediators for metastasis, while current therapies targeting MMPs are still suboptimal. Lan et al. demonstrated that the combination of Asiatic acid and Naringenin effectively suppressed metastasis of melanoma and lung carcinoma via inhibiting TGF-β-induced MMP2 transcription, post-translational activation, and function, which may represent a promising anti-metastatic therapy.

Kallistatin is a multiple functional serine protease inhibitor that protects against vascular injury, organ damage and tumor progression. Kallistatin treatment reduces inflammation and fibrosis in the progression of chronic kidney disease (CKD), but the molecular mechanisms underlying this protective process and whether kallistatin plays an endogenous role are incompletely understood. In the present study, we observed that renal kallistatin levels were significantly lower in patients with CKD. It was also positively correlated with estimated glomerular filtration rate (eGFR) and negatively correlated with serum creatinine level. Unilateral ureteral obstruction (UUO) in animals also led to down-regulation of kallistatin protein in the kidney, and depletion of endogenous kallistatin by antibody injection resulted in aggravated renal fibrosis, which was accompanied by enhanced Wnt/β-catenin activation. Conversely, overexpression of kallistatin attenuated renal inflammation, interstitial fibroblast activation and tubular injury in UUO mice. The protective effect of kallistatin was due to the suppression of TGF-β and β-catenin signaling pathways and subsequent inhibition of epithelial-to-mesenchymal transition (EMT) in cultured tubular cells. In addition, kallistatin could inhibit TGF-β-mediated fibroblast activation via modulation of Wnt4/β-catenin signaling pathway. Therefore, endogenous kallistatin protects against renal fibrosis by modulating Wnt/β-catenin-mediated EMT and fibroblast activation. Down-regulation of kallistatin in the progression of renal fibrosis underlies its potential as a valuable clinical biomarker and therapeutic target in CKD.

Increasing clinical evidence shows that acute kidney injury (AKI) is a common and severe complication in critically ill COVID-19 patients. The older age, the severity of COVID-19 infection, the ethnicity, and the history of smoking, diabetes, hypertension, and cardiovascular disease are the risk factor for AKI in COVID-19 patients. Of them, inflammation may be a key player in the pathogenesis of AKI in patients with COVID-19. It is highly possible that SARS-COV-2 infection may trigger the activation of multiple inflammatory pathways including angiotensin II, cytokine storm such as interleukin-6 (IL-6), C-reactive protein (CRP), TGF-β signaling, complement activation, and lung-kidney crosstalk to cause AKI. Thus, treatments by targeting these inflammatory molecules and pathways with a monoclonal antibody against IL-6 (Tocilizumab), C3 inhibitor AMY-101, anti-C5 antibody, anti-TGF-β OT-101, and the use of CRRT in critically ill patients may represent as novel and specific therapies for AKI in COVID-19 patients.

Diabetic nephropathy (DN) is a major cause of end-stage renal disease, but treatment remains ineffective. C-reactive protein (CRP) is pathogenic in DN, which significantly correlated with dipeptidyl peptidase-4 (DPP4) expression in diabetic patients with unknown reason. Here, using our unique CRP(tg)-db/db mice, we observed human CRP markedly induced renal DPP4 associated with enhanced kidney injury compared with db/db mice. Interestingly, linagliptin, a US Food and Drug Administration (FDA)-approved specific DPP4 inhibitor, effectively blocked this CRP-driven DN in the CRP(tg)-db/db mice. Mechanistically, CRP evoked DPP4 in cultured renal tubular epithelial cells, where CD32b/nuclear factor κB (NF-κB) signaling markedly enriched p65 binding on the DPP4 promoter region to increase its transcription. Unexpectedly, we further discovered that CRP triggers dimerization of DPP4 with CD32b at protein level, forming a novel DPP4/CD32b/NF-κB signaling circuit for promoting CRP-mediated DN. More importantly, linagliptin effectively blocked the circuit, thereby inhibiting the CRP/CD32b/NF-κB-driven renal inflammation and fibrosis. Thus, DPP4 may represent a precise druggable target for CRP-driven DN.

Rationale: Transforming Growth Factor-beta (TGF-β) /Smad3 signaling has been shown to play important roles in fibrotic and inflammatory diseases, but its role in beta cell function and type 2 diabetes is unknown. Methods: The role of Smad3 in beta cell function under type 2 diabetes condition was investigated by genetically deleting Smad3 from db/db mice. Phenotypic changes of pancreatic islets and beta cell function were compared between Smad3 knockout db/db (Smad3KO-db/db) mice and Smad3 wild-type db/db (Smad3WT-db/db) mice, and other littermate controls. Islet-specific RNA-sequencing was performed to identify Smad3-dependent differentially expressed genes associated with type 2 diabetes. In vitro beta cell proliferation assay and insulin secretion assay were carried out to validate the mechanism by which Smad3 regulates beta cell proliferation and function. Results: The results showed that Smad3 deficiency completely protected against diabetes-associated beta cell loss and dysfunction in db/db mice. By islet-specific RNA-sequencing, we identified 8160 Smad3-dependent differentially expressed genes associated with type 2 diabetes, where Smad3 deficiency markedly prevented the down-regulation of those genes. Mechanistically, Smad3 deficiency preserved the expression of beta cell development mediator Pax6 in islet, thereby enhancing beta cell proliferation and function in db/db mice in vivo and in Min6 cells in vitro. Conclusions: Taken together, we discovered a pathogenic role of Smad3 in beta cell loss and dysfunction via targeting the protective Pax6. Thus, Smad3 may represent as a novel therapeutic target for type 2 diabetes prevention and treatment.

Transforming growth factor β (TGF-β) is a well-known key mediator for the progression and metastasis of lung carcinoma. However, cost-effective anti-TGF-β therapeutics for lung cancer remain to be explored. Specifically, the low efficacy in drug delivery greatly limits the clinical application of small molecular inhibitors of TGF-β. In the present study, specific inhibitor of Smad3 (SIS3) is developed into a self-carried nanodrug (SCND-SIS3) using the reprecipitation method, which largely improves its solubility and bioavailability while reduces its nephrotoxicity. Compared to unmodified-SIS3, SCND-SIS3 demonstrates better anti-cancer effects through inducing tumor cell apoptosis, inhibiting angiogenesis, and boosting NK cell-mediated immune responses in syngeneic Lewis Lung Cancer (LLC) mouse model. Better still, it could achieve comparable anti-cancer effect with just one-fifth the dose of unmodified-SIS3. Mechanistically, RNA-sequencing analysis and cytokine array results unveil a TGF-β/Smad3-dependent immunoregulatory landscape in NK cells. In particular, SCND-SIS3 promotes NK cell cytotoxicity by ameliorating Smad3-mediated transcriptional inhibition of Ndrg1. Furthermore, improved NK cell cytotoxicity by SCND-SIS3 is associated with higher expression of activation receptor Nkp46, and suppressed levels of Trib3 and TSP1 as compared with unmodified-SIS3. Taken together, SCND-SIS3 possesses superior anti-cancer effects with enhanced bioavailability and biocompatibility, therefore representing as a novel therapeutic strategy for lung carcinoma with promising clinical potential.

Smad3 deficiency prevents the development of type 2 diabetic nephropathy; however, the underlying molecular mechanisms remain unknown. In this study, we aimed to identify Smad3‐related genes involved in the pathogenesis of diabetic kidney disease. High‐throughput RNA sequencing was performed to profile the whole transcriptome in the diabetic kidney of Smad3 WT‐db/db, Smad3 KO‐db/db, Smad3+/− db/db and their littermate control db/m mice at 20 weeks. The gene ontology, pathways and alternative splicing of differentially expressed protein‐coding genes and long non‐coding RNAs related to Smad3 in diabetic kidney were analysed. Compared to Smad3 WT‐db/db mice, Smad3 KO‐db/db mice exhibited an alteration of genes associated with RNA splicing and metabolism, whereas heterozygosity deletion of Smad3 (Smad3+/− db/db mice) significantly altered genes related to cell division and cell cycle. Notably, three protein‐coding genes (Upk1b, Psca and Gdf15) and two lncRNAs (NONMMUG023520.2 and NONMMUG032975.2) were identified to be Smad3‐dependent and to be associated with the development of diabetic nephropathy. By using whole transcriptome RNA sequencing, we identified novel Smad3 transcripts related to the development of diabetic nephropathy. Thus, targeting these transcripts may represent a novel and effective therapy for diabetic nephropathy.

Angiotensin‐converting enzyme‐2 (ACE2) and Mas receptor are the major components of the ACE2/Ang 1‐7/Mas axis and have been shown to play a protective role in hypertension and hypertensive nephropathy individually. However, the effects of dual deficiency of ACE2 and Mas (ACE2/Mas) on Ang II‐induced hypertensive nephropathy remain unexplored, which was investigated in this study in a mouse model of hypertension induced in either ACE2 knockout (KO) or Mas KO mice and in double ACE2/Mas KO mice by subcutaneously chronic infusion of Ang II. Compared with wild‐type (WT) animals, mice lacking either ACE2 or Mas significantly increased blood pressure over 7‐28 days following a chronic Ang II infusion (P

Transforming growth factor β (TGF-β)/Smad3 signaling plays a central role in chronic heart disease. Here, we report that targeting Smad3 with a Smad3 inhibitor SIS3 in an established mouse model of hypertension significantly improved cardiac dysfunctions by preserving the left ventricle (LV) ejection fraction (LVEF) and LV fractional shortening (LVFS), while reducing the LV mass. In addition, SIS3 treatment also halted the progression of myocardial fibrosis by blocking α-smooth muscle actin-positive (α-SMA+) myofibroblasts and collagen matrix accumulation, and inhibited cardiac inflammation by suppressing interleukin (IL)-1β, tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein 1 (MCP1), intercellular cell adhesion molecule-1 (ICAM1) expression, and infiltration of CD3+ T cells and F4/80+ macrophages. Interestingly, treatment with SIS3 did not alter levels of high blood pressure, revealing a blood pressure-independent cardioprotective effect of SIS3. Mechanistically, treatment with SIS3 not only directly inactivated TGF-β/Smad3 signaling but also protected cardiac Smad7 from Smurf2-mediated proteasomal ubiquitin degradation. Because Smad7 functions as an inhibitor for both TGF-β/Smad and nuclear factor κB (NF-κB) signaling, increased cardiac Smad7 could be another mechanism through which SIS3 treatment blocked Smad3-mediated myocardial fibrosis and NF-κB-driven cardiac inflammation. In conclusion, SIS3 is a therapeutic agent for hypertensive heart disease. Results from this study demonstrate that targeting Smad3 signaling with SIS3 may be a novel and effective therapy for chronic heart disease., Graphical Abstract, Meng and colleagues developed a novel and effective therapy for hypertensive heart disease by specifically targeting Smad3, a key mediator of downstream TGF-β signaling, with a Smad3 inhibitor SIS3. They found that SIS3 treatment significantly improved cardiac dysfunctions and suppressed myocardial fibrosis and inflammation in an established mouse model of hypertension.

Diabetic nephropathy (DN) is a major cause of end-stage kidney disease, where TGF-β1/Smad signaling plays an important role in the disease progression. Our previous studies demonstrated a combination of Traditional Chinese Medicine derived Smad7 agonist Asiatic Acid (AA) and Smad3 inhibitor Naringenin (NG), AANG, effectively suppressed the progression of renal fibrosis

Macrophage‐Myofibroblast Transition In article number 2101235 Philip Chiu‐Tsun Tang, Patrick Ming‐Kuen Tang, Hui‐Yao Lan, and co‐workers discovered that tumor‐associated macrophages are capable for de novo generating pathogenic cancer‐associated fibroblasts via a direct mechanism macrophage‐myofibroblast transition (MMT), representing a novel therapeutic target in the tumor microenvironment of solid cancers. Here, the macrophages undergoing MMT were coexpressing CAF (α‐SMA, red) and macrophage marker (F4/80, green) with nuclei staining (blue), visualized by immunofluorescence with an experimental Lewis lung carcinoma (LLC) model. [Image: see text]

Cancer‐associated fibroblasts (CAFs) are important in tumor microenvironment (TME) driven cancer progression. However, CAFs are heterogeneous and still largely underdefined, better understanding their origins will identify new therapeutic strategies for cancer. Here, the authors discovered a new role of macrophage‐myofibroblast transition (MMT) in cancer for de novo generating protumoral CAFs by resolving the transcriptome dynamics of tumor‐associated macrophages (TAM) with single‐cell resolution. MMT cells (MMTs) are observed in non‐small‐cell lung carcinoma (NSCLC) associated with CAF abundance and patient mortality. By fate‐mapping study, RNA velocity, and pseudotime analysis, existence of novel macrophage‐lineage‐derived CAF subset in the TME of Lewis lung carcinoma (LLC) model is confirmed, which is directly transited via MMT from M2‐TAM in vivo and bone‐marrow‐derived macrophages (BMDM) in vitro. Adoptive transfer of BMDM‐derived MMTs markedly promote CAF formation in LLC‐bearing mice. Mechanistically, a Smad3‐centric regulatory network is upregulated in the MMTs of NSCLC, where chromatin immunoprecipitation sequencing(ChIP‐seq) detects a significant enrichment of Smad3 binding on fibroblast differentiation genes in the macrophage‐lineage cells in LLC‐tumor. More importantly, macrophage‐specific deletion and pharmaceutical inhibition of Smad3 effectively block MMT, therefore, suppressing the CAF formation and cancer progression in vivo. Thus, MMT may represent a novel therapeutic target of CAF for cancer immunotherapy., Here, the authors discovered a direct mechanism of tumor‐associated macrophages for de novo generating protumoral cancer‐associated fibroblasts (CAF) via macrophage‐myofibroblast transition (MMT) by resolving their transcriptome dynamics at single‐cell resolution. Encouragingly, targeting the MMT key regulator Smad3 effectively blocked the production and protumoral actions of CAF, evidencing its translational potential as a novel therapeutic target in the tumour microenvironment for cancer.

Transforming growth factor-β/Smad3 signaling plays an important role in diabetic nephropathy, but its underlying working mechanism remains largely unexplored. The current study uncovered the pathogenic role and underlying mechanism of a novel Smad3-dependent long noncoding RNA (lncRNA) (LRNA9884) in type 2 diabetic nephropathy (T2DN). We found that LRNA9884 was significantly upregulated in the diabetic kidney of db/db mice at the age of 8 weeks preceding the onset of microalbuminuria and was associated with the progression of diabetic renal injury. LRNA9884 was induced by advanced glycation end products and tightly regulated by Smad3, and its levels were significantly blunted in db/db mice and cells lacking Smad3. More importantly, kidney-specific silencing of LRNA9884 effectively attenuated diabetic kidney injury in db/db mice, as shown by the reduction of histological injury, albuminuria excretion, and serum creatinine. Mechanistically, we identified that LRNA9884 promoted renal inflammation-driven T2DN by triggering MCP-1 production at the transcriptional level, and its direct binding significantly enhanced the promoter activity of MCP-1. Thus, LRNA9884 is a novel Smad3-dependent lncRNA that is highly expressed in db/db mice associated with T2DN development. Targeting of LRNA9884 effectively blocked MCP-1–dependent renal inflammation, therefore suppressing the progressive diabetic renal injury in db/db mice. This study reveals that LRNA9884 may be a novel and precision therapeutic target for T2DN in the future.

Renal fibrosis is one of the most characterized pathological features in chronic kidney disease (CKD). Progressive fibrosis eventually leads to renal failure, leaving dialysis or allograft transplantation the only clinical option for CKD patients. Transforming growth factor-β (TGF-β) is the key mediator in renal fibrosis and is an essential regulator for renal inflammation. Therefore, the general blockade of the pro-fibrotic TGF-β may reduce fibrosis but may risk promoting renal inflammation and other side effects due to the diverse role of TGF-β in kidney diseases. Long non-coding RNAs (lncRNAs) are RNA transcripts with more than 200 nucleotides and have been regarded as promising therapeutic targets for many diseases. This review focuses on the importance of TGF-β and lncRNAs in renal inflammation, fibrogenesis, and the potential applications of TGF-β and lncRNAs as the therapeutic targets and biomarkers in renal fibrosis and CKD are highlighted.

Diabetic cardiomyopathy (DCM) is a common diabetic complication characterized by diastolic relaxation abnormalities, myocardial fibrosis and chronic heart failure. Although TGF‐β/Smad3 signalling has been shown to play a critical role in chronic heart disease, the role and mechanisms of Smad3 in DCM remain unclear. We reported here the potential role of Smad3 in the development of DCM by genetically deleting the Smad3 gene from db/db mice. At the age of 32 weeks, Smad3WT‐db/db mice developed moderate to severe DCM as demonstrated by a marked increase in the left ventricular (LV) mass, a significant fall in the LV ejection fraction (EF) and LV fractional shortening (FS), and progressive myocardial fibrosis and inflammation. In contrast, db/db mice lacking Smad3 (Smad3KO‐db/db) were protected against the development of DCM with normal cardiac function and undetectable myocardial inflammation and fibrosis. Interestingly, db/db mice with deleting one copy of Smad3 (Smad3 ± db/db) did not show any cardioprotective effects. Mechanistically, we found that deletion of Smad3 from db/db mice largely protected cardiac Smad7 from Smurf2‐mediated ubiquitin proteasome degradation, thereby inducing IBα to suppress NF‐kB‐driven cardiac inflammation. In addition, deletion of Smad3 also altered Smad3‐dependent miRNAs by up‐regulating cardiac miR‐29b while suppressing miR‐21 to exhibit the cardioprotective effect on Smad3KO‐db/db mice. In conclusion, results from this study reveal that Smad3 is a key mediator in the pathogenesis of DCM. Targeting Smad3 may be a novel therapy for DCM.

Plasma levels of neuropeptide Y (NPY) are elevated in patients with acute myocardial infarction (AMI), but its role in AMI remains unclear, which was examined here in NPY wild-type/knockout (WT/KO) mice treated with/without exogenous NPY and its Y1 receptor antagonist (Y1Ra) BIBP 3226. We found that AMI mice lacking NPY developed more severe AMI than WT mice with worse cardiac dysfunction, progressive cardiac inflammation and fibrosis, and excessive apoptosis but impairing angiogenesis. All of these changes were reversed when the NPY KO mice were treated with exogenous NPY in a dose-dependent manner. Interestingly, treatment with NPY also dose dependently attenuated AMI in WT mice, which was blocked by BIBP 3226. Phenotypically, cardiac NPY was de novo expressed by infiltrating macrophages during the repairing or fibrosing process in heart-failure patients and AMI mice. Mechanistically, NPY was induced by transforming growth factor (TGF)-β1 in bone marrow-derived macrophages and signaled through its Y1R to exert its pathophysiological activities by inhibiting p38/nuclear factor κB (NF-κB)-mediated M1 macrophage activation while promoting the reparative M2 phenotype in vivo and in vitro. In conclusion, NPY can attenuate AMI in mice. Inhibition of cardiac inflammation and fibrosis while enhancing angiogenesis but reducing apoptosis may be the underlying mechanisms through which NPY attenuates cardiac remodeling and deterioration of function following AMI.

Diabetic kidney disease (DKD) is the most common diabetic complication and is a leading cause of end-stage kidney disease. Increasing evidence shows that DKD is regulated not only by many classical signaling pathways but also by epigenetic mechanisms involving chromatin histone modifications, DNA methylation, and non-coding RNA (ncRNAs). In this review, we focus on our current understanding of the role and mechanisms of ncRNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) in the pathogenesis of DKD. Of them, the regulatory role of TGF-β/Smad3-dependent miRNAs and lncRNAs in DKD is highlighted. Importantly, miRNAs and lncRNAs as biomarkers and therapeutic targets for DKD are also described, and the perspective of ncRNAs as a novel therapeutic approach for combating diabetic nephropathy is also discussed.