Inhibition of Macrophage Recruitment to Heart Valves Mediated by the C-C Chemokine Receptor Type 2 Attenuates Valvular Inflammation Induced by Group A Streptococcus in Lewis Rats.

BACKGROUND: Rheumatic heart disease (RHD) is an autoimmune disease caused by recurrent infections of Group A streptococcus (GAS), ultimately leading to inflammation and the fibrosis of heart valves. Recent studies have highlighted the crucial role of C-C chemokine receptor type 2-positive (CCR2+) macrophages in autoimmune diseases and tissue fibrosis. However, the specific involvement of CCR2+ macrophages in RHD remains unclear. METHODS: This study established an RHD rat model using inactivated GAS and complete Freund's adjuvant, demonstrating a correlation between CCR2+ macrophages and fibrosis in the mitral valves of these rats. RESULTS: Intraperitoneal injection of the CCR2 antagonist Rs-504393 significantly reduced macrophage infiltration, inflammation, and fibrosis in valve tissues of RHD rats compared to the solvent-treated group . Existing evidence suggests that C-C motif chemokine ligand 2 (CCL2) acts as the primary recruiting factor for CCR2+ cells. To validate this, human monocytic leukemia cells (THP-1) were cultured in vitro to assess the impact of recombinant CCL2 protein on macrophages. CCL2 exhibited pro-inflammatory effects similar to lipopolysaccharide (LPS), promoting M1 polarization in macrophages. Moreover, the combined effect of LPS and CCL2 was more potent than either alone. Knocking down CCR2 expression in THP-1 cells using small interfering RNA suppressed the pro-inflammatory response and M1 polarization induced by CCL2. CONCLUSIONS: The findings from this study indicate that CCR2+ macrophages are pivotal in the valvular remodeling process of RHD. Targeting the CCL2/CCR2 signaling pathway may therefore represent a promising therapeutic strategy to alleviate valve fibrosis in RHD.

Potential Involvement of M1 Macrophage and VLA4/VCAM-1 Pathway in the Valvular Damage Due to Rheumatic Heart Disease.

BACKGROUND: Rheumatic heart disease (RHD) is caused by inflammatory cells mistakenly attacking the heart valve due to Group A Streptococcus (GAS) infection, but it is still unclear which cells or genes are involved in the process of inflammatory cells infiltrating the valve. Inflammatory infiltration into the target tissue requires an increase in the expression of phosphorylated vascular endothelial-cadherin (p-VE-cad), p-VE-cad can increase the endothelial permeability and promote the migration of inflammatory cells across the endothelium. P-VE-cad is potentially regulated by RAS-related C3 botulinum substrate 1 (RAC1), together with phosphorylated proline-rich tyrosine kinase 2 (p-PYK2). While RAC1/p-PYK2/p-VE-cad is triggered by the activation of vascular cell adhesion molecule-1 (VCAM-1). VCAM-1 is related to M1 macrophages adhering to the endothelium via very late antigen 4 (VLA4). Inflammatory infiltration into the valve is extremely important in the early pathogenesis of RHD. However, there is no relevant research on whether M1/VLA4/VCAM-1/RAC1/p-PYK2/p-VE-cad is involved in RHD; therefore, what we explored in this study was whether M1/VLA4/VCAM-1/RAC1/p-PYK2/p-VE-cad is involved. METHODS: We established a rat model of RHD and a cell model of M1 macrophage and endothelial cell cocultivation. Subsequently, we measured the degree of inflammatory cell infiltration, the levels of IL-6/IL-17, the degree of fibrosis (COL3/1), and the expression levels of fibrosis markers (FSP1, COL1A1 and COL3A1) in the heart valves of RHD rats. Additionally, we detected the expression of M1/M2 macrophage biomarkers in rat model and cell model, as well as the expression of M1/VLA4/VCAM-1/RAC1/p-PYK2/p-VE-cad. We also tested the changes in endothelial permeability after coculturing M1 macrophages and endothelial cells. RESULTS: Compared to those in the control group, the levels of inflammatory cell infiltration and fibrotic factors in the heart valves of RHD rats were significantly higher; the expression of M1 macrophage biomarkers (iNOS, CD86 and TNF-α) in RHD rats was significantly higher; and significantly higher than the expression of M2 macrophage biomarkers (Arg1 and TGF-ß). And the expression levels of VLA4/VCAM-1 and RAC1/p-PYK2/p-VE-cad in the hearts of RHD rats were significantly higher. At the cellular level, after coculturing M1 macrophages with endothelial cells, the expression levels of VLA4/VCAM-1 and RAC1/p-PYK2/p-VE-cad were significantly higher, and the permeability of the endothelium was significantly greater due to cocultivation with M1 macrophages. CONCLUSIONS: All the results suggested that M1 macrophages and the VLA4/VCAM-1 pathway are potentially involved in the process of inflammatory infiltration in RHD.

Profibrotic VEGFR3-Dependent Lymphatic Vessel Growth in Autoimmune Valvular Carditis.

BACKGROUND: Rheumatic heart disease is the major cause of valvular heart disease in developing nations. Endothelial cells (ECs) are considered crucial contributors to rheumatic heart disease, but greater insight into their roles in disease progression is needed. METHODS: We used a Cdh5-driven EC lineage-tracing approach to identify and track ECs in the K/B.g7 model of autoimmune valvular carditis. Single-cell RNA sequencing was used to characterize the EC populations in control and inflamed mitral valves. Immunostaining and conventional histology were used to evaluate lineage tracing and validate single-cell RNA-sequencing findings. The effects of VEGFR3 (vascular endothelial growth factor receptor 3) and VEGF-C (vascular endothelial growth factor C) inhibitors were tested in vivo. The functional impact of mitral valve disease in the K/B.g7 mouse was evaluated using echocardiography. Finally, to translate our findings, we analyzed valves from human patients with rheumatic heart disease undergoing mitral valve replacements. RESULTS: Lineage tracing in K/B.g7 mice revealed new capillary lymphatic vessels arising from valve surface ECs during the progression of disease in K/B.g7 mice. Unsupervised clustering of mitral valve single-cell RNA-sequencing data revealed novel lymphatic valve ECs that express a transcriptional profile distinct from other valve EC populations including the recently identified PROX1 (Prospero homeobox protein 1)+ lymphatic valve ECs. During disease progression, these newly identified lymphatic valve ECs expand and upregulate a profibrotic transcriptional profile. Inhibiting VEGFR3 through multiple approaches prevented expansion of this mitral valve lymphatic network. Echocardiography demonstrated that K/B.g7 mice have left ventricular dysfunction and mitral valve stenosis. Valve lymphatic density increased with age in K/B.g7 mice and correlated with worsened ventricular dysfunction. Importantly, human rheumatic valves contained similar lymphatics in greater numbers than nonrheumatic controls. CONCLUSIONS: These studies reveal a novel mode of inflammation-associated, VEGFR3-dependent postnatal lymphangiogenesis in murine autoimmune valvular carditis, with similarities to human rheumatic heart disease.

Bilateral Secondary Femorocele in a Case of Ascites Due to Cardiac Cirrhosis Following Dual Valvular Replacement for Rheumatic Heart Disease.

Background: Fluid collection in a femoral hernia sac designated as a femorocele is an extremely uncommon surgical condition. Till date 9 cases of unilateral femorocele and one case of bilateral femorocele have been reported in English literature. Objective: Thus making the case presented the second case of bilateral femorocele in English literature. Case report: A case of bilateral femorocele in a patient suffering from rheumatic heat disease who had undergone dual valvular replacement with ascites due to cardiac cirrhosis is presented to highlight the surgical challenges in management of such a rare case. Discussion: Pathophysiology, clinical features, investigations and managemeny of femorocele are discussed. Conclusion: Contrast enhanced CT scan of the abdomen and scrotum is diagnostic. Open surgery in the form of dissection of sac with high ligation followed by obliteration of femoral ring is therapeutic. There is no scope of laparoscopy in such a case.

Sentinel Lymph Node Mapping and Staging Surgery Via Gasless Transvaginal Natural Orifice Transluminal Endoscopic Surgery: A Case Report of an Endometrial Cancer Patient and Comorbid Rheumatic Heart Disease.

BACKGROUND Conventional laparoscopic surgery and transvaginal natural orifice transluminal endoscopic surgery (vNOTES) both use CO2 pneumoperitoneum to expose the surgical space. However, CO2 pneumoperitoneum is undoubtedly dangerous for patients with rheumatic heart disease (RHD) and can cause cardiopulmonary impairments. Therefore, we selected the sentinel lymph node (SLN) mapping strategy to guide the staging surgery via gasless vNOTES for an endometrial cancer (EC)-patient with comorbid RHD. Here, we discuss whether our selected surgical method was safe and feasible for this patient. CASE REPORT A 43-year-old woman with a history of RHD, severe mitral regurgitation, and pulmonary hypertension for more than 30 years received diagnostic curettage for irregular vaginal bleeding for more than 1 month. Pathological examinations revealed the occurrence of highly differentiated intrauterine endometrioid adenocarcinoma. She was admitted to the gynecological ward of our hospital for further surgery. We performed EC staging surgery with SLN mapping via gasless vNOTES and adopted a series of effective measures to solve the intraoperative complications of surgical space exposure. Surgery was successful. The patient recovered well and was discharged 5 days after surgery. She has been followed up in the gynecological clinic for nearly 1 year. At the time of this report, she had good recovery, no recurrence and metastasis, and normal tumor markers. CONCLUSIONS For EC patients with comorbid RHD pathology, application of staging surgery with SLN mapping via gasless vNOTES was shown to be safe and feasible. This approach is expected to be highly effective for patients with contraindications to CO2 pneumoperitoneum laparoscopy.

Histopathological evaluation of chronic rheumatic mitral valve stenosis: the association with clinical presentation, pathogenesis, and management at a National Cardiac Institute, Tanzania.

AIMS: The histopathology of mitral valve (MV) tissues have been reported in necropsy and retrospective studies. We prospectively studied the histopathological changes in rheumatic mitral stenosis using advanced techniques and corroborated these with clinical presentation, pathogenesis, and management. METHODS: From January 2020 to February 2021, surgically excised rheumatic stenotic MV from 54 Tanzanian patients were studied. These were examined using hematoxylin-eosin, von Kossa staining, and immunohistochemistry. RESULTS: The median (range) age of patients was 39 (14-57) years with 34 (63%) females. Secondary prophylaxis was given to 7 (13%) patients and 2 (3.7%) had evidence of rheumatic fever (RF). With hematoxylin-eosin, 37 (68.5%) specimens showed fibrinoid degeneration (FD), 44 (81.5%) leucocytic infiltrates, 6 (11.1%) Aschoff nodules, 30 (55.6%) calcification, and 39 (72.2%) fibrosis. Thirty-five (64.8%) specimens were positive to von Kossa. The proportion of specimens positive for CD3, CD20, CD68, and CD8 were 46 (85.2%), 35 (64.8%), 39 (72.2%), and 8 (14.8%) respectively. Valvular calcium was high among older patients, males and with a higher trans-MV gradient. The degree of inflammatory cellular infiltration was associated with valvular calcification, FD with ARF, leucocytic infiltrates with disease duration of <10 years, and fibrosis with the absence of atrial fibrillation. C-reactive protein and anti-streptolysin titres were high in CD20 and CD8 staining cells. CONCLUSION: This study confirms that high MV calcium are found in patients who are old, male, and with severe mitral stenosis. The association between clinical parameters with histopathological-immunohistochemical studies observed in our study provides new insight to disease presentation. We found a low rate of secondary prophylaxis and two patients with ARF. Our findings are comparable with those from other countries suggesting similar pathogenesis and thus intervention modalities. This is the first study on mitral valve histopathology to be reported from Africa.

Effect of Sox9 on TGF-ß1-mediated atrial fibrosis.

Atrial fibrosis is a crucial mechanism responsible for atrial fibrillation (AF). Sex-determining region Y-box containing gene 9 (Sox9) plays a pivotal role in fibrosis of many organs such as the skin, kidney, and liver. However, there are few studies about the occurrence and maintenance of Sox9 in atrial fibrosis. In this study, we investigated the role of Sox9 in the fibrotic phenotype of human atrial tissues and rat atrial fibroblasts in vitro. In the human right atrial tissue, Masson's trichrome staining, immunofluorescence, real-time quantitative polymerase chain reaction, and western blot analysis were carried out to explore the relationship between Sox9 and atrial fibrosis at the morphological, functional, and molecular levels. In cultured atrial fibroblasts, Sox9 was overexpressed by adenovirus or depleted by siRNA, and then, recombinant human transforming growth factor (TGF)-ß1 was added. Immunofluorescence analysis, western blot analysis, Transwell assay, and scratch assay were used to analyze the cells. In patient atrial tissues, Sox9 was increased with worsened atrial fibrosis, and this increase was related to AF severity. In rat atrial fibroblasts, Sox9 was promoted by TGF-ß1, and the α-smooth muscle actin (α-SMA) protein level and the ability of cell migration were increased after Sox9 overexpression by adenovirus, while the α-SMA protein level and the cell migration ability were decreased after Sox9 depletion by siRNA. In conclusion, Sox9 is involved in the regulation of fibrosis in the atria and may be located downstream of TGF-ß1. Our findings may provide a new perspective to treat atrial fibrosis during AF.

Interference with the expression of S1PR1 or STAT3 attenuates valvular damage due to rheumatic heart disease.

Rheumatic heart disease (RHD) affects numerous individuals annually; however, its pathogenesis remains unclear. The sphingosine 1­phosphate receptor 1 (S1PR1) and signal transducer and activator of transcription 3 (STAT3) have recently been shown to be involved in valvular damage via the promotion of the differentiation of T helper 17 (Th17) cells during the development of RHD­induced valvular damage. The present study investigated whether altering the expression of S1PR1 or STAT3 attenuates valvular damage due to RHD. Inactivated group A streptococcus (GAS) was used to establish a rat model of RHD. Recombinant adeno­associated viral vectors carrying an S1PR1 overexpression sequence were used to overexpress S1PR1. STAT3 small interfering RNA (STAT3­siRNA) was used to inhibit STAT3 expression. Reverse transcription­quantitative PCR (RT­qPCR) was performed to detect the mRNA expression of S1PR1, STAT3, collagen type III α1 chain (Col3a1) and fibroblast­specific protein 1. Western blotting (WB) and immunohistochemistry were used to detect the levels of S1PR1, STAT3, phosphorylated (p­) STAT3, and retinoic acid­related orphan receptor Î³T (RORγt) proteins. Enzyme­linked immunosorbent assays (ELISAs) and immunohistochemistry were used to detect the levels of interleukin (IL)­6 and IL­17. Hematoxylin and eosin (H&E) staining and Sirius Red staining were performed to evaluate the degree of inflammation and fibrosis in the valvular tissues. S1PR1 expression was decreased in the valvular tissues of the rats with RHD. The levels of IL­6, IL­17 and p­STAT3 in the rats with RHD were increased. The degree of valvular inflammation and fibrosis in the rats with RHD was also increased. The overexpression of S1PR1 and the inhibition of STAT3 reduced the total p­STAT3 level, resulting in decreased levels of IL­6, IL­17 and RORγt, and a reduced degree of valvular inflammation and fibrosis. These results suggest that the expression of S1PR1 and STAT3 may be involved in valvular tissue damage due to RHD. Thus, strategies designed to interfere with the expression of S1PR1 or STAT3 may affect the expression of Th17 cell­related cytokines and may thus attenuate valvular damage due to RHD.

miRNA-1183-targeted regulation of Bcl-2 contributes to the pathogenesis of rheumatic heart disease.

To determine whether up-regulation of miR-1183 targeting the gene for anti-apoptotic factor, B-cell lymphoma 2 (BCL-2) contributes to apoptosis in patients with rheumatic heart disease (RHD). Peripheral blood samples were isolated for miR-1183 characterization. The function of miRNA-1183 in RHD using miRNA mimic on PBMCs and THP-1 cell models. The binding of miR-1183 and Bcl-2 gene was confirmed by luciferase activity test. We also measured expression levels of BCL-2 in heart valve tissue from patients with RHD using ELISA and immunohistochemistry. In silico analysis and reporter gene assays indicated that miR-1183 directly targets the mRNA encoding BCL-2. It is found that miR-1183 binds directly to the 3'UTR of the BCL-2 mRNA and down-regulates the mRNA and protein levels of BCL-2. Overexpression of miR-1183 in RHD patients and cell lines down-regulated BCL-2 expression and induced apoptosis. With the progression of the disease, the expression of BCL-2 in the heart valve tissue of patients with RHD decreased. MiRNA-1183 is up-regulated in RHD and induces cardiac myocyte apoptosis through direct targeting and suppression of BCL-2, both of which might play important roles in RHD pathogenesis. During the compensatory period of RHD, up-regulated miR-1183 destroyed the balance of apoptosis proteins (Bax and BAK) in Bcl-2 family, enhance the apoptosis cascade reaction and reduce the anti apoptosis effect. The significantly higher expression levels of miR-1183 appear to play distinct roles in RHD pathogenesis by regulation BCL-2, possibly affecting myocardial apoptosis and remodeling in the context of RHD.

Clinicopathological Bird's-Eye View of Left Atrial Myocardial Fibrosis in 121 Patients With Persistent Atrial Fibrillation: Developing Architecture and Main Cellular Players.

BACKGROUND: Scientific research on atrial fibrosis in atrial fibrillation (AF) has mainly focused on quantitative or molecular features. The purpose of this study was to perform a clinicoarchitectural/structural investigation of fibrosis to provide one key to understanding the electrophysiological/clinical aspects of AF. METHODS: We characterized the fibrosis (amount, architecture, cellular components, and ultrastructure) in left atrial biopsies from 121 patients with persistent/long-lasting persistent AF (group 1; 59 males; 60±11 years; 91 mitral disease-related AF, 30 nonmitral disease-related AF) and from 39 patients in sinus rhythm with mitral valve regurgitation (group 2; 32 males; 59±12 years). Ten autopsy hearts served as controls. RESULTS: Qualitatively, the fibrosis exhibited the same characteristics in all cases and displayed particular architectural scenarios (which we arbitrarily subdivided into 4 stages) ranging from isolated foci to confluent sclerotic areas. The percentage of fibrosis was larger and at a more advanced stage in group 1 versus group 2 and, within group 1, in patients with rheumatic disease versus nonrheumatic cases. In patients with AF with mitral disease and no rheumatic disease, the percentage of fibrosis and the fibrosis stages correlated with both left atrial volume index and AF duration. The fibrotic areas mainly consisted of type I collagen with only a minor cellular component (especially fibroblasts/myofibroblasts; average value range 69-150 cells/mm2, depending on the areas in AF biopsies). A few fibrocytes-circulating and bone marrow-derived mesenchymal cells-were also detectable. The fibrosis-entrapped cardiomyocytes showed sarcolemmal damage and connexin 43 redistribution/internalization. CONCLUSIONS: Atrial fibrosis is an evolving and inhomogeneous histological/architectural change that progresses through different stages ranging from isolated foci to confluent sclerotic zones which-seemingly-constrain impulse conduction across restricted regions of electrotonically coupled cardiomyocytes. The fibrotic areas mainly consist of type I collagen extracellular matrix and, only to a lesser extent, mesenchymal cells.

Genetic variants in rheumatic fever and rheumatic heart disease.

Genetic association studies in rheumatic heart disease (RHD) have the potential to contribute toward our understanding of the pathogenetic mechanism, and may shed light on controversies about RHD etiology. Furthermore, genetic association studies may uncover biomarkers that can be used to identify susceptible individuals, and contribute toward developing vaccine and novel therapeutic targets. Genetic predisposition to rheumatic fever and RHD has been hypothesized by findings from familial studies and observed associations between genes located in the human leukocyte antigens on chromosome 6p21.3 and elsewhere in the genome. We sought to summarize, from published Genetic association studies in RHD, evidence on genetic variants implicated in RHD susceptibility. Using HuGENet™ systematic review methods, we evaluated 66 studies reporting on 42 genes. Existing meta-analyses of candidate gene studies suggest that TGF-ß1 [rs1800469], and IL-1ß [rs2853550] single nucleotide polymorphisms (SNPs) contribute to susceptibility to RHD, whereas the TNF-α [rs1800629 and rs361525], TGF-ß1 [rs1800470 and rs4803457], IL-6 [rs1800795], IL-10 [rs1800896] were not associated with RHD. However, candidate gene studies in RF/RHD are relatively small, thus lacking statistical power to identify reliable and reproducible findings, emphasizing the need for large-scale multicenter studies with different populations.

Inhibition of miR­155­5p attenuates the valvular damage induced by rheumatic heart disease.

Autoimmunity is involved in the valvular damage caused by rheumatic heart disease (RHD). Increased evidence has linked microRNAs (miRNAs/miRs) to autoimmune disease. Signal transducer and activator of transcription 3 (STAT3) and sphingosine­1­phosphate receptor 1 (S1PR1) and suppressor of cytokine signaling 1 (SOCS1) have been widely studied for their roles in autoimmunity and inflammation. Thus, the current study aims to investigate the role played by miR­155­5p in RHD­induced valvular damage via the S1PR1, SOCS1/STAT3 and interleukin (IL)­6/STAT3 signaling pathways. An RHD rat model was induced by inactivated Group A streptococci and complete Freund's adjuvant. A recombinant adeno­associated virus (AAV­miR155­inhibitor) was used to inhibit the expression of miR­155­5p in the heart. Inflammation and fibrosis were assessed by hematoxylin and eosin staining and Sirius red staining. The expression of miR­155­5p in valvular tissues and serum exosomes was detected by reverse transcription­quantitative PCR. S1PR1, SOCS1, STAT3, phosphorylated STAT3, IL­6 and IL­17 protein expression was detected by western blotting and immunohistochemistry. The relationships between miR­155­5p and S1PR1 and SOCS1 were detected by dual luciferase assays. Cytokine concentrations were measured by ELISA. The expression of miR­155­5p in valve tissues and serum exosomes was increased along with decreased S1PR1 and activated SOCS1/STAT3 signaling in the RHD model. The expression of IL­6 and IL­17 was increased in the valves and the serum. Dual luciferase assays showed that miR­155­5p directly targeted S1PR1 and SOCS1. Inhibition of valvular miR­155­5p through AAV pretreatment increased S1PR1 expression and inhibited activation of the SOCS1/STAT3 signal pathway as a result of attenuated valvular inflammation and fibrosis as well as a decrease in IL­6 and IL­17 in the valves and serum. These results suggest that inhibition of miR­155­5p can reduce RHD­induced valvular damage via the S1PR1, SOCS1/STAT3 and IL­6/STAT3 signaling pathways.

Prevalence and progression of rheumatic heart disease: a global systematic review and meta-analysis of population-based echocardiographic studies.

This systematic review and meta-analysis aimed to provide a contemporaneous estimate of the global burden of rheumatic heart disease (RHD) from echocardiographic population-based studies. We searched multiple databases between January 01, 1996 and October 17, 2017. Random-effect meta-analysis was used to pool data. We included 82 studies (1,090,792 participant) reporting data on the prevalence of RHD and 9 studies on the evolution of RHD lesions. The pooled prevalence of RHD was 26.1‰ (95%CI 19.2-33.1) and 11.3‰ (95%CI 7.2-16.2) for studies which used the World Heart Federation (WHF) and World Health Organization (WHO) criteria, respectively. The prevalence of RHD varied inversely with the level of a country's income, was lower with the WHO criteria compared to the WHF criteria, and was lowest in South East Asia. Definite RHD progressed in 7.5% (95% CI 1.5-17.6) of the cases, while 60.7% (95% CI 42.4-77.5) of cases remained stable over the course of follow-up. The proportion of cases borderline RHD who progressed to definite RHD was 11.3% (95% CI 6.9-16.5). The prevalence of RHD across WHO regions remains high. The highest prevalence of RHD was noted among studies which used the WHF diagnostic criteria. Definite RHD tends to progress or remain stable over time.

TGFß1 and HGF regulate CTGF expression in human atrial fibroblasts and are involved in atrial remodelling in patients with rheumatic heart disease.

OBJECTIVE: This study aimed to investigate the effects of transforming growth factor ß1 (TGF ß1) and hepatocyte growth factor (HGF) on the expression of connective tissue growth factor (CTGF) in human atrial fibroblasts, and to explore the relationship of these factors in atrial fibrosis and atrial anatomical remodelling (AAR) of patients with atrial fibrillation (AF). METHODS: Fresh right auricular appendix tissue of 20 patients with rheumatic heart disease undergoing valve replacement surgery was collected during surgeries, 10 patients had sinus rhythm(SR), and 10 patients had chronic atrial fibrillation (CAF). Atrial fibroblasts were then cultured from the tissues with differential attachment technique and treated with either TGFß1 (10 ng/mL) or HGF (100 ng/mL). CTGF mRNA levels were measured by RT-PCR, and CTGF protein content was determined using immunofluorescence and Western blotting assays. RESULTS: CAF group had higher left atrial diameters (LADs) and higher CTGF mRNA expression in atrial fibroblasts compared with SR group. The CTGF protein content in CAF group was higher than that of SR group and positively correlated with LAD and AF duration. After CAF group was treated with TGFß1, CTGF mRNA and protein expression were significantly down-regulated, whereas when treated with HGF, expression was up-regulated compared with SR group. CONCLUSIONS: Increased CTGF expression was associated with enlarged LAD, atrial fibrosis and AAR in patients with AF. TGFß1 and HGF regulate CTGF expression in human atrial fibroblasts with up-regulation of mRNA and down-regulation of protein, therefore, either promote or inhibit atrial fibrosis, which could be related to the incidence and persistence of AF.

Association between late gadolinium enhancement and global longitudinal strain in patients with rheumatic mitral stenosis.

The correlation between the extent of myocardial fibrosis and subclinical left ventricle (LV) systolic dysfunction in rheumatic mitral stenosis (MS) has not been widely studied. We sought to evaluate the correlation between the extent of LV myocardial fibrosis quantified by late gadolinium enhancement (LGE) using cardiac magnetic resonance (CMR) and global longitudinal strain (GLS) by speckle tracking echocardiography (STE) in patients with rheumatic MS. We prospectively evaluated 36 consecutive rheumatic MS patients who were planning to undergo mitral valve surgery. Then we evaluate the correlation between the extent of LV myocardial fibrosis quantified by LGE CMR and the systolic LV function by GLS using STE. Thirty-six patients with mean age of 45.7 ± 9.9 years old, showed mean LGE was 4.9 ± 2.7%. The mean LV ejection fraction (EF) measured by CMR was 50 ± 10.8%, and the mean LV GLS was 13.5 ± 3.9%. There was a moderate correlation between GLS and LGE (r - 0.432, p = 0.009). There were no correlations between GLS with mitral valve area (MVA) with r 0.149, p = 0.385, mean mitral valve gradient (MVG) with r -0.078, p = 0.653, and LVEF (r 0.299, p = 0.076). There was a moderate correlation between LGE and GLS in patients with rheumatic MS.

Rheumatoid nodule on the anterior mitral valve leaflet.

Symptomatic cardiac rheumatoid nodules are a rare but recognized manifestation of rheumatoid arthritis. We describe the surgical management of a rheumatic nodule involving the anterior leaflet of the mitral valve.

Assessment of myocardial fibrosis by late gadolinium enhancement imaging and biomarkers of collagen metabolism in chronic rheumatic mitral regurgitation.

BACKGROUND: In chronic rheumatic mitral regurgitation (CRMR), involvement of the myocardium in the rheumatic process has been controversial. Therefore, we sought to study the presence of fibrosis using late gadolinium enhancement cardiac magnetic resonance imaging (LGE-CMR) and biomarkers of collagen turnover in CRMR. METHODS: Twenty-two patients with CRMR underwent CMR and echocardiography. Serum concentrations of matrix metalloproteinase- 1 (MMP-1), tissue inhibitor of MMP-1 (TIMP- 1), MMP-1-to-TIMP-1 ratio, procollagen III N-terminal pro-peptide (PIIINP) and procollagen type IC peptide (PIP) were measured. RESULTS: Four patients had fibrosis on LGE-CMR. PICP and PIIINP concentrations were similar to those of the controls, however MMP-1 concentration was increased compared to that of the controls (log MMP-1 3.5 ± 0.7 vs 2.7 ± 0.9, p = 0.02). There was increased MMP-1 activity as the MMP-1-to- TIMP-1 ratio was higher in CRMR patients compared to the controls ( -1.2 ± 0.6 vs -2.1 ± 0.89, p = 0.002). CONCLUSIONS: Myocardial fibrosis was rare in CRMR patients. CRMR is likely a disease characterised by the predominance of collagen degradation rather than increased synthesis and myocardial fibrosis.

CD301b/MGL2+ Mononuclear Phagocytes Orchestrate Autoimmune Cardiac Valve Inflammation and Fibrosis.

BACKGROUND: Valvular heart disease is common and affects the mitral valve (MV) most frequently. Despite the prevalence of MV disease (MVD), the cellular and molecular pathways that initiate and perpetuate it are not well understood. METHODS: K/B.g7 T-cell receptor transgenic mice spontaneously develop systemic autoantibody-associated autoimmunity, leading to fully penetrant fibroinflammatory MVD and arthritis. We used multiparameter flow cytometry, intracellular cytokine staining, and immunofluorescent staining to characterize the cells in inflamed K/B.g7 MVs. We used genetic approaches to study the contribution of mononuclear phagocytes (MNPs) to MVD in this model. Specifically, we generated K/B.g7 mice in which either CX3CR1 or CD301b/macrophage galactose N-acetylgalactosamine-specific lectin 2 (MGL2)-expressing MNPs were ablated. Using K/B.g7 mice expressing Cx3Cr1-Cre, we conditionally deleted critical inflammatory molecules from MNPs, including the Fc-receptor signal-transducing tyrosine kinase Syk and the cell adhesion molecule very late antigen-4. We performed complementary studies using monoclonal antibodies to block key inflammatory molecules. We generated bone marrow chimeric mice to define the origin of the inflammatory cells present in the MV and to determine which valve cells respond to the proinflammatory cytokine tumor necrosis factor (TNF). Finally, we examined specimens from patients with rheumatic heart disease to correlate our findings to human pathology. RESULTS: MNPs comprised the vast majority of MV-infiltrating cells; these MNPs expressed CX3CR1 and CD301b/MGL2. Analogous cells were present in human rheumatic heart disease valves. K/B.g7 mice lacking CX3CR1 or in which CD301b/MGL2-expressing MNPs were ablated were protected from MVD. The valve-infiltrating CD301b/MGL2+ MNPs expressed tissue-reparative molecules including arginase-1 and resistin-like molecule α. These MNPs also expressed the proinflammatory cytokines TNF and interleukin-6, and antibody blockade of these cytokines prevented MVD. Deleting Syk from CX3CR1-expressing MNPs reduced their TNF and interleukin-6 production and also prevented MVD. TNF acted through TNF receptor-1 expressed on valve-resident cells to increase the expression of vascular cell adhesion molecule-1. Conditionally deleting the vascular cell adhesion molecule-1 ligand very late antigen-4 from CX3CR1-expressing MNPs prevented MVD. CONCLUSIONS: CD301b/MGL2+ MNPs are key drivers of autoimmune MVD in K/B.g7 mice and are also present in human rheumatic heart disease. We define key inflammatory molecules that drive MVD in this model, including Syk, TNF, interleukin-6, very late antigen-4, and vascular cell adhesion molecule-1.

Low-level overexpression of p53 promotes warfarin-induced calcification of porcine aortic valve interstitial cells by activating Slug gene transcription.

The most frequently used oral anti-coagulant warfarin has been implicated in inducing calcification of aortic valve interstitial cells (AVICs), whereas the mechanism is not fully understood. The low-level activation of p53 is found to be involved in osteogenic transdifferentiation and calcification of AVICs. Whether p53 participates in warfarin-induced AVIC calcification remains unknown. In this study, we investigated the role of low-level p53 overexpression in warfarin-induced porcine AVIC (pAVIC) calcification. Immunostaining, quantitative PCR, and Western blotting revealed that p53 was expressed in human and pAVICs and that p53 expression was slightly increased in calcific human aortic valves compared with non-calcific valves. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining indicated that apoptosis slightly increased in calcific aortic valves than in non-calcific valves. Warfarin treatment led to a low-level increase of p53 mRNA and protein in both pAVICs and mouse aortic valves. Low-level overexpression of p53 in pAVICs via an adenovirus vector did not affect pAVIC apoptosis but promoted warfarin-induced calcium deposition and expression of osteogenic markers. shRNA-mediated p53 knockdown attenuated the pAVIC calcium deposition and osteogenic marker expression. Moreover, ChIP and luciferase assays showed that p53 was recruited to the slug promoter and activated slug expression in calcific pAVICs. Of note, overexpression of Slug increased osteogenic marker Runx2 expression, but not pAVIC calcium deposition, and Slug knockdown attenuated pAVIC calcification and p53-mediated pAVIC calcium deposition and expression of osteogenic markers. In conclusion, we found that p53 plays an important role in warfarin induced pAVIC calcification, and increased slug transcription by p53 is required for p53-mediated pAVIC calcification.

Henoch-Schonlein purpura, post-streptococcal glomerulonephritis and acute rheumatic carditis after Group A ß-haemolytic streptococcal infection.

Besides association with acute rheumatic fever (ARF) and acute glomerulonephritis (APSGN), in up to 40% of cases, Group A ß-haemolytic streptococcal (GABHS) infections are also implicated as a trigger for Henoch-Schonlein purpura (HSP). A 7-year-old girl with GABHS throat infection who developed HSP, APSGN and rheumatic carditis is reported. She presented with palpable purpura and arthritis in both ankles and later developed carditis characterised by mitral/aortic regurgitation and glomerulonephritis characterised by mixed nephritic/nephrotic syndrome. She had a raised anti-streptolysin titre (ASOT), blood urea nitrogen and creatinine and hypocomplementaemia (C3), and renal biopsy demonstrated endocapillary and extracapillary proliferative glomerulonephritis with crescents. Immunofluorescence microscopy demonstrated a 'full house' of immunoglobulin and complement, viz. IgA + 2, IgG + 3, IgM + 2, C3c + 1, Clq + 2 with predominantly IgG deposition. One week earlier, her 4-year-old sister had presented to another hospital with HSP complicated by microscopic haematuria, nephrotic-range proteinuria and gastro-intestinal involvement, and with raised ASOT and low C3 levels. Although HSP has been associated with either ARF or APSGN, this is the first case of a child with HSP, ARF and APSGN in combination.