Exploring personalized treatment for cardiac graft rejection based on a four-archetype analysis model and bioinformatics analysis.

Heart transplantation is the gold standard for treating patients with advanced heart failure. Although improvements in immunosuppressive therapies have significantly reduced the frequency of cardiac graft rejection, the incidences of T cell-mediated rejection (TCMR) and antibody-mediated rejection remain almost unchanged. A four-archetype analysis (4AA) model, developed by Philip F. Halloran, illustrated this problem well. It provided a new dimension to improve the accuracy of diagnoses and an independent system for recalibrating the histology guidelines. However, this model was based on the invasive method of endocardial biopsy, which undoubtedly increased the postoperative risk of heart transplant patients. Currently, little is known regarding the associated genes and specific functions of the different phenotypes. We performed bioinformatics analysis (using machine-learning methods and the WGCNA algorithm) to screen for hub-specific genes related to different phenotypes, based Gene Expression Omnibus accession number GSE124897. More immune cell infiltration was observed with the ABMR, TCMR, and injury phenotypes than with the stable phenotype. Hub-specific genes for each of the four archetypes were verified successfully using an external test set (accession number GSE2596). Logistic-regression models based on TCMR-specific hub genes and common hub genes were constructed with accurate diagnostic utility (area under the curve > 0.95). RELA, NFKB1, and SOX14 were identified as transcription factors important for TCMR/injury phenotypes and common genes, respectively. Additionally, 11 Food and Drug Administration-approved drugs were chosen from the DrugBank Database for each four-archetype model. Tyrosine kinase inhibitors may be a promising new option for transplant rejection treatment. KRAS signaling in cardiac transplant rejection is worth further investigation. Our results showed that heart transplant rejection subtypes can be accurately diagnosed by detecting expression of the corresponding specific genes, thereby enabling precise treatment or medication.

HTRA1-driven detachment of type I collagen from endoplasmic reticulum contributes to myocardial fibrosis in dilated cardiomyopathy.

BACKGROUND: The aberrant secretion and excessive deposition of type I collagen (Col1) are important factors in the pathogenesis of myocardial fibrosis in dilated cardiomyopathy (DCM). However, the precise molecular mechanisms underlying the synthesis and secretion of Col1 remain unclear. METHODS AND RESULTS: RNA-sequencing analysis revealed an increased HtrA serine peptidase 1 (HTRA1) expression in patients with DCM, which is strongly correlated with myocardial fibrosis. Consistent findings were observed in both human and mouse tissues by immunoblotting, quantitative reverse transcription polymerase chain reaction (qRT-PCR), immunohistochemistry, and immunofluorescence analyses. Pearson's analysis showed a markedly positive correlation between HTRA1 level and myocardial fibrosis indicators, including extracellular volume fraction (ECV), native T1, and late gadolinium enhancement (LGE), in patients with DCM. In vitro experiments showed that the suppression of HTRA1 inhibited the conversion of cardiac fibroblasts into myofibroblasts and decreased Col1 secretion. Further investigations identified the role of HTRA1 in promoting the formation of endoplasmic reticulum (ER) exit sites, which facilitated the transportation of Col1 from the ER to the Golgi apparatus, thereby increasing its secretion. Conversely, HTRA1 knockdown impeded the retention of Col1 in the ER, triggering ER stress and subsequent induction of ER autophagy to degrade misfolded Col1 and maintain ER homeostasis. In vivo experiments using adeno-associated virus-serotype 9-shHTRA1-green fluorescent protein (AAV9-shHTRA1-GFP) showed that HTRA1 knockdown effectively suppressed myocardial fibrosis and improved left ventricular function in mice with DCM. CONCLUSIONS: The findings of this study provide valuable insights regarding the treatment of DCM-associated myocardial fibrosis and highlight the therapeutic potential of targeting HTRA1-mediated collagen secretion.

TREM2 Insufficiency Protects against Pulmonary Fibrosis by Inhibiting M2 Macrophage Polarization.

Rationale Idiopathic pulmonary fibrosis (IPF) is a lung disease with high mortality, limited treatment options and an unknown aetiology. M2 macrophages play a critical role in the pathological process of IPF. Triggering receptor expressed on myeloid cells-2 (TREM2) participates in the regulation of macrophages, although its role in IPF remains elusive. METHODS: This study examined the role of TREM2 in macrophage regulation using a well-established bleomycin (BLM)-induced pulmonary fibrosis (PF) mouse model. TREM2 insufficiency was induced by intratracheal treatment with TREM2-specific siRNA. The effects of TREM2 on IPF were evaluated using histological staining and molecular biological methods. RESULTS: TREM2 expression levels were significantly elevated in the lungs of IPF patients and mice with BLM-induced pulmonary fibrosis mice. Bioinformatics analysis revealed that IPF patients with higher TREM2 expression had a shorter survival time, and that TREM2 expression was closely associated with fibroblasts and M2 macrophages. Gene Ontology (GO) enrichment analysis showed that found TREM2-related differentially expressed genes (DEGs) were associated with inflammatory responses, extracellular matrix (ECM) and collagen formation. Single-cell RNA sequencing analysis revealed that TREM2 was predominantly expressed in macrophages. TREM2 insufficiency inhibited BLM-induced pulmonary fibrosis and M2 macrophage polarization. Mechanistic studies showed that TREM2 insufficiency suppressed the activation of STAT6 and the expression of fibrotic factors such as Fibronectin (Fib), Collagen I (Col I) and α- smooth muscle actin (α-SMA). CONCLUSION: Our study showed that TREM2 insufficiency might alleviate pulmonary fibrosis possibly through macrophage polarization regulation via STAT6 activation, providing a promising macrophage-related approach for the clinical therapy of pulmonary fibrosis.

SWAP70 Overexpression Protects Against Pathological Cardiac Hypertrophy in a TAK1-Dependent Manner.

Background Pathological cardiac hypertrophy is regarded as a critical precursor and independent risk factor of heart failure, and its inhibition prevents the progression of heart failure. Switch-associated protein 70 (SWAP70) is confirmed important in immunoregulation, cell maturation, and cell transformation. However, its role in pathological cardiac hypertrophy remains unclear. Methods and Results The effects of SWAP70 on pathological cardiac hypertrophy were investigated in Swap70 knockout mice and Swap70 overexpression/knockdown cardiomyocytes. Bioinformatic analysis combined with multiple molecular biological methodologies were adopted to elucidate the mechanisms underlying the effects of SWAP70 on pathological cardiac hypertrophy. Results showed that SWAP70 protein levels were significantly increased in failing human heart tissues, experimental transverse aortic constriction-induced mouse hypertrophic hearts, and phenylephrine-stimulated isolated primary cardiomyocytes. Intriguingly, phenylephrine treatment decreased the lysosomal degradation of SWAP70 by disrupting the interaction of SWAP70 with granulin precursor. In vitro and in vivo experiments revealed that Swap70 knockdown/knockout accelerated the progression of pathological cardiac hypertrophy, while Swap70 overexpression restrained the cardiomyocyte hypertrophy. SWAP70 restrained the binding of transforming growth factor ß-activated kinase 1 (TAK1) and TAK1 binding protein 1, thus blocking the phosphorylation of TAK1 and downstream c-Jun N-terminal kinase/P38 signaling. TAK1 interacted with the N-terminals (1-192) of SWAP70. Swap70 (193-585) overexpression failed to inhibit cardiac hypertrophy when the TAK1-SWAP70 interaction was disrupted. Either inhibiting the phosphorylation or suppressing the expression of TAK1 rescued the exaggerated cardiac hypertrophy induced by Swap70 knockdown. Conclusions SWAP70 suppressed the progression of cardiac hypertrophy, possibly by inhibiting the mitogen-activated protein kinases signaling pathway in a TAK1-dependent manner, and lysosomes are involved in the regulation of SWAP70 expression level.

Switch-associated protein 70 protects against nonalcoholic fatty liver disease through suppression of TAK1.

BACKGROUND AND AIMS: NAFLD is a progressive disease without known effective drug treatments. Switch-associated protein 70 (SWAP70) is a guanine nucleotide exchange factor that participates in the regulation of many cellular processes. However, the role of SWAP70 in NAFLD remains unclear. This study aimed to identify the function and mechanism of SWAP70 in NAFLD. APPROACH AND RESULTS: The results showed that the expression of SWAP70 was significantly increased in mice and hepatocytes after metabolic stimulation. Overexpression of SWAP70 in hepatocytes suppressed lipid deposition and inflammation, and SWAP70 knockdown created the inverse effect. Using hepatocyte-specific Swap70 knockout and overexpression mice fed a high-fat, high-cholesterol diet, we demonstrated that SWAP70 suppressed the progression of nonalcoholic steatohepatitis by inhibiting lipid accumulation, inflammatory response, and fibrosis. Mechanically, RNA sequencing analysis and immunoprecipitation assays revealed that SWAP70 inhibited the interaction between transforming growth factor ß-activated kinase 1 (TAK1) binding protein 1 and TAK1 and sequentially suppressed the phosphorylation of TAK1 and subsequent c-Jun N-terminal kinase/P38 signaling. Inhibition of TAK1 activation blocked hepatocyte lipid deposition and inflammation caused by SWAP70 knockdown. CONCLUSIONS: SWAP70 is a protective molecule that can suppress the progression of NAFLD by inhibiting hepatic steatosis and inflammation. SWAP70 may be important for mitigating the progression of NAFLD.

M2a macrophage can rescue proliferation and gene expression of benign prostate hyperplasia epithelial and stroma cells from insulin-like growth factor 1 knockdown.

BACKGROUND: Benign prostatic hyperplasia (BPH) is a common disease in elderly men and is often accompanied by chronic inflammation. Macrophages (several subtypes) are the main inflammatory cells that infiltrate the hyperplastic prostate and are found to secrete cytokines and growth factors. The current study aims to explore the effect of M2a macrophages on the development of BPH via insulin-like growth factor 1 (IGF-1). METHODS: Human prostate tissues, prostate, and monocyte cell lines (WPMY-1, BPH-1, and THP-1) were used. THP-1 was polarized into several subtypes with cytokines. The expression and localization of IGF-1 and M2 macrophages were determined via immunofluorescent staining, quantitative real-time polymerase chain reaction, and Western blot analysis. Flow cytometry and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays were used to investigate the effects of different subtypes of macrophages on prostate cells. IGF-1 in WPMY-1 and BPH-1 cells was silenced and cocultured with or without M2a macrophages. Cell proliferation, apoptosis, cell cycle, epithelial-mesenchymal transition (EMT), and fibrosis processes were examined. RESULTS: The polarized subtypes of macrophages were verified by amplifying their specific markers. M2a macrophages enhanced prostate cell proliferation more significantly and CD206 was more expressed in hyperplastic prostate. IGF-1 was localized in both epithelial and stromal components of prostate and upregulated in BPH tissues. M2a macrophages expressed more IGF-1 than other subtypes. Knockdown of IGF-1 in WPMY-1 and BPH-1 cells attenuated cell proliferation, promoted cell apoptosis, retarded cell cycle at the G0/G1 phase, and suppressed the EMT process in BPH-1 cells as well as the fibrotic process in WPMY-1 cells, which was reversible when cocultured with M2a macrophages. CONCLUSION: These data demonstrated that knockdown of IGF-1 expression in cultured BPH epithelial and stromal cells reduces proliferation and increases apoptosis. These effects are reversed by coculture with M2a macrophages.

Changes in the expression and functional activities of Myosin II isoforms in human hyperplastic prostate.

Benign prostatic hyperplasia (BPH) is a common disease among aging males with the etiology remaining unclear. We recently found myosin II was abundantly expressed in rat and cultured human prostate cells with permissive roles in the dynamic and static components. The present study aimed to explore the expression and functional activities of myosin II isoforms including smooth muscle (SM) myosin II (SMM II) and non-muscle myosin II (NMM II) in the hyperplastic prostate. Human prostate cell lines and tissues from normal human and BPH patients were used. Hematoxylin and Eosin (H&E), Masson's trichrome, immunohistochemical staining, in vitro organ bath, RT-polymerase chain reaction (PCR) and Western-blotting were performed. We further created cell models with NMM II isoforms silenced and proliferation, cycle, and apoptosis of prostate cells were determined by cell counting kit-8 (CCK-8) assay and flow cytometry. Hyperplastic prostate SM expressed more SM1 and LC17b isoforms compared with their alternatively spliced counterparts, favoring a slower more tonic-type contraction and greater force generation. For BPH group, blebbistatin (BLEB, a selective myosin II inhibitor), exhibited a stronger effect on relaxing phenylephrine (PE) pre-contracted prostate strips and inhibiting PE-induced contraction. Additionally, NMMHC-A and NMMHC-B were up-regulated in hyperplastic prostate with no change in NMMHC-C. Knockdown of NMMHC-A or NMMHC-B inhibited prostate cell proliferation and induced apoptosis, with no changes in cell cycle. Our novel data demonstrate that expression and functional activities of myosin II isoforms are altered in human hyperplastic prostate, suggesting a new pathological mechanism for BPH. Thus, the myosin II system may provide potential new therapeutic targets for BPH/lower urinary tract symptoms (LUTS).

Changes in the expression and function of the PDE5 pathway in the obstructed urinary bladder.

Our study aims to explore changes in bladder contractility and the phosphodiesterase type 5 (PDE5) signalling pathway in response to partial bladder outlet obstruction (PBOO). A surgically induced male rat PBOO model and human obstructed bladder tissues were used. Histological changes were examined by H&E and Masson's trichrome staining. Bladder strip contractility was measured via organ bath. The expressions of nitric oxide synthase (NOS) isoforms, PDE5, muscarinic cholinergic receptor (CHRM) isoforms and PDE4 isoforms in bladder were detected by RT-PCR and Western blotting. The immunolocalization of the PDE5 protein and its functional activity were also determined. PBOO bladder tissue exhibited significant SM hypertrophy and elevated responsiveness to KCl depolarization and the muscarinic receptor agonist carbachol. NOS isoforms, PDE5, CHRM2, CHRM3 and PDE4A were up-regulated in obstructed bladder tissue, whereas no change in PDE4B and PDE4D isoform expression was observed. With regard to PDE5, it was expressed in the SM bundles of bladder. Interestingly, obstructed bladder exhibited less relaxation responsiveness to sodium nitroprusside (SNP), but an exaggerated PDE5 inhibition effect. The up-regulation of PDE5 could contribute to the lack of effect on Qmax for benign prostatic hyperplasia/lower urinary tract symptom (BPH/LUTS) patients treated with PDE5 inhibitors. Moreover, PDE5 (with presence of NO) and PDE4 may serve as new therapeutic targets for bladder diseases such as BPH-induced LUTS and overactive bladder (OAB).