Abstract
Aims
Degenerative mitral valve dystrophy (MVD) leading to mitral valve prolapse is the most frequent form of MV disease, and there is currently no pharmacological treatment available. The ...limited understanding of the pathophysiological mechanisms leading to MVD limits our ability to identify therapeutic targets. This study aimed to reveal the main pathophysiological pathways involved in MVD via the multimodality imaging and transcriptomic analysis of the new and unique knock-in (KI) rat model for the FilaminA-P637Q (FlnA-P637Q) mutation associated-MVD.
Methods and results
Wild-type (WT) and KI rats were evaluated morphologically, functionally, and histologically between 3-week-old and 3-to-6-month-old based on Doppler echocardiography, 3D micro-computed tomography (microCT), and standard histology. RNA-sequencing and Assay for Transposase-Accessible Chromatin (ATAC-seq) were performed on 3-week-old WT and KI mitral valves and valvular cells, respectively, to highlight the main signalling pathways associated with MVD. Echocardiographic exploration confirmed MV elongation (2.0 ± 0.1 mm vs. 1.8 ± 0.1, P = 0.001), as well as MV thickening and prolapse in KI animals compared to WT at 3 weeks. 3D MV volume quantified by microCT was significantly increased in KI animals (+58% vs. WT, P = 0.02). Histological analyses revealed a myxomatous remodelling in KI MV characterized by proteoglycans accumulation. A persistent phenotype was observed in adult KI rats. Signalling pathways related to extracellular matrix homeostasis, response to molecular stress, epithelial cell migration, endothelial to mesenchymal transition, chemotaxis and immune cell migration, were identified based on RNA-seq analysis. ATAC-seq analysis points to the critical role of transforming growth factor-β and inflammation in the disease.
Conclusion
The KI FlnA-P637Q rat model mimics human myxomatous MVD, offering a unique opportunity to decipher pathophysiological mechanisms related to this disease. Extracellular matrix organization, epithelial cell migration, response to mechanical stress, and a central contribution of immune cells are highlighted as the main signalling pathways leading to myxomatous MVD. Our findings pave the road to decipher underlying molecular mechanisms and the specific role of distinct cell populations in this context.
Graphical Abstract
Graphical Abstract
Leptin receptor (ObR-b) is overexpressed in pulmonary artery smooth muscle cells (PA-SMCs) from patients with pulmonary arterial hypertension (PAH) and is implicated in both mechanisms that ...contribute to pulmonary vascular remodeling: hyperproliferation and inflammation. Our aim was to investigate the role of ubiquitin-specific peptidase 8 (USP8) in ObR-b overexpression in PAH.
We performed in situ and in vitro experiments in human lung specimens and isolated PA-SMCs combined with 2 different in vivo models in rodents and we generated a mouse with an inducible USP8 deletion specifically in smooth muscles.
Our results showed an upregulation of USP8 in the smooth muscle layer of distal pulmonary arteries from patients with PAH, and upregulation of USP8 expression in PAH PA-SMCs, compared to controls. USP8 inhibition in PAH PA-SMCs significantly blocked both ObR-b protein expression level at the cell surface as well as ObR-b-dependant intracellular signaling pathway as shown by a significant decrease in pSTAT3 expression. USP8 was required for ObR-b activation in PA-SMCs and its inhibition prevented Ob-mediated cell proliferation through STAT3 pathway. USP8 inhibition by the chemical inhibitor DUBs-IN-2 protected against the development of experimental PH in the 2 established experimental models of PH. Targeting USP8 specifically in smooth muscle cells in a transgenic mouse model also protected against the development of experimental PH.
Our findings highlight the role of USP8 in ObR-b overexpression and pulmonary vascular remodeling in PAH.
Calcific aortic valve stenosis (CAVS) affects more than 10% of the population over 75 years old and aortic valve replacement is the only option. Biological prostheses are largely used in this context ...but have a limited durability with the development of structural bioprosthetic degeneration (SVD) post implantation. Lipid factors such as proprotein convertase subtilisin/kexin type 9 (PCSK9), Lipoprotein (a) and LDL-cholesterol have been recently associated with SVD, even if the underlying mechanisms remain unknown.
We aim at evaluating the impact of hypercholesterolemia and/or PCSK9 on the early processes leading to SVD.
To study bioprosthetic tissue degradation, subcutaneous implantation of this tissue was performed in wild type (WT), PCSK9 knock-out (KO) and PCSK9 overexpressing mice for 28 days. A qualitative anatomopathological score based on cell density, infiltration, and matrix degradation was developed. Immunochemistry was performed to characterize immune cells infiltration, with a focus on anti- (i.e. CD163+) and pro-inflammatory (i.e. F4/80+) macrophages, lymphocytes (CD3+) and polynuclear eosinophils (i.e. EPX+).
Histological analysis of explanted punches revealed an infiltration of mononuclear cells into the biological matrix among the 3 groups, with a significant increase in mice overexpressing PCSK9 compared to WT and KO. The anatomopathological score was significantly higher in overexpressing mice compared to the WT and KO mice (median value 7.0 4.1–8.4 versus 4.0 3.0–4.5 and 2.8 2.0–3.9, respectively; P=0.008). The macrophages (both CD163+ and F4/80+) and polynuclear eosinophils were the most abundant cell types, even if few lymphocytes were also observed. Interestingly, polynuclear eosinophils were more abundant in mice overexpressing PCSK9 compared to WT and KO (P=0.01 and P=0.002, respectively).
Hypercholesterolemia and/or high circulating PCSK9 level enhance the response against the bioprosthetic tissue. Macrophages are the most abundant cell types invading the tissue. Hypercholesterolemia/high PCSK9 potentiate the infiltration of polynuclear eosinophils, which point out for a direct impact on the inflammatory response post implantation. Deeper molecular analyses will provide mechanistic evidence linking hypercholesterolemia and PCSK9 to the early inflammation processes leading to SVD.
Mitral valve prolapse is a common disease affecting 2 to 3% of the population, characterized by a myxomatous mitral valve disease (MVD). In 2007, the first causal mutation in the FLNA gene ...(FLNA-P637Q), was associated with MVD. The FlnA-P637Q KI rat model was recently generated. As early as 3 weeks (D21), the presence of MVD was confirmed and was associated with transcriptomic signature of chemotaxis and immune cells migration.
This study aims at delineating the role of the recruitment and activation of macrophages in the pathophysiology of MVD.
KI and WT animals were phenotyped at birth (D0), day 2 (D2), and day 7 (D7). Morphological alterations of the MV were evaluated by anatomopathological score (D7). Cell proportions were analyzed by flow cytometry (D7 and D21), and molecular phenotyping was done by bulk RNAseq (D7). WT and KI MVs were also dissociated for valvular interstitial cells (VICs) primary culture and subsequent in vitro studies.
Flow cytometry at D21 revealed a 2-fold increase proportion of myeloid cells in KI MV compared to WT (13 vs. 7%). Interestingly, although no morphological differences were seen at D0 an D2, we observed a remodeling of the MV characteristic of the presence of MVD at D7. Accordingly, histological score was significantly higher in KI rats (7 vs. 4, P<0.001). This was consistent with the transcriptomic analysis of D7 MV that revealed enrichment of GO-Terms (P<0.05) related to ECM-receptor interaction, cell adhesion, proteoglycans and extracellular space. Of note, pathways related to chemotaxis were also enriched (P<0.05), but flow cytometry at D7 revealed no difference in myeloid cell proportions (7% in WT and KI). The in vitro modulation of mechanical stresses imposed to VICs (i.e. stiffness or laminar flow) induced an increased expression of monocytes adhesion molecules ICAM1 and GITRL in KI compared to WT VICs (all P<0.05).
MVD is observed from day 7 in FLNA-KI rats and is associated to pro-inflammatory signature enrichment but no increase in macropahges proportions. The response of VICs to in vitro mechanical stresses suggest that FlnA-KI VICs are more susceptible to express monocytes adhesion molecules which could explain the increased proportion of macrophages seen at D21 in FlnA-KI rats.
Myxomatous valve dystrophy (MVD) is characterized by an elongated, and thickened mitral valve (MV) with extracellular matrix (ECM) accumulation. Using the FilaminA P637Q Knock-In (FLNA-KI) rat model ...of MVD, we previously demonstrated that it recapitulates the human pathology. RNA-Seq analysis revealed important contribution of immune cells. Cytometry experiments showed increase proportion of macrophages at D21 in KI valves.
The objective is to decipher how macrophages contribute to the development of MVD.
Seven days, two days and newborn FLNA-KI rats (D7, D2 and D0) were phenotyped through a quantitative histological analysis. Cytometry, to assess valvular macrophages population, as well as qPCR for expression of the key molecular targets was performed.
At D7, FLNA-KI rats exhibit thickened and elongated mitral valve leaflets, and presented a higher histological score as compared to WT rats (P=0.0007). qPCR experiments revealed dysregulation of genes involved in ECM regulation (HAS1 FC=8 and HYAL1 FC=0.63 vs. WT, both P<0.01) and endothelial dysfunction (ESM1 FC=2.2 vs. WT, P=0.005) concordant with the results at D21. Preliminary results at D0 and D2 showed upregulation of genes related to ECM remodeling (HAS1 FC=2.6 and 3.2 vs. WT, respectively, P<0.01). Morphological analysis at D0 and D2 is still ongoing. Although a pro-inflammatory environment was present at D7 (CCL7 FC=2.6 and S100A8 FC=4.2 vs. WT, P<0.5), no changes in the proportion of macrophages was detected by cytometry (6% in both WT and KI MV, P>0.5). The expression of inflammatory cytokines was not different at D0 and D2 in KI rats’ mitral valves (all P>0.5).
Our study indicates that MVD is present in KI as soon as birth. The ECM remodeling is probably a basis for the pro-inflammatory environment that promotes macrophages recruitment in the valvular leaflets. This suggests that macrophages infiltration is not causal of MVD but rather participates to the evolution of the disease. Further investigations are ongoing to understand how macrophages contribute to the pathophysiology of MVD.
Calcific aortic valve stenosis (CAVS) is one of the most frequent cardiovascular diseases affecting more than 10% of the population over 70years old. There is currently no pharmacological treatment ...to stop this process, and the only solution remains to replace the diseased valve with a prosthesis. A shift toward the use of biological prosthesis occurred in the last 10years, as these protheses presented better hemodynamic profile without lifelong anticoagulation treatment. However, the main disadvantage of the bioprostheses is their limited durability overtime, with the development of a gradual structural bioprosthetic degeneration (SVD) in the following years post intervention.
Our objective is to test the role of hypercholesterolemia and/or PCSK9, a well-know modulator of the cholesterol metabolism, on the early process leading to SVD.
As the currently gold standard technic to test calcification potential of biological tissue from bioprosthetic valve, we implanted subcutaneously 5mm punches of biological matrix from bovine pericardium, in 3 groups of mice: PCSK9 KO, WT and overexpressing hepatic PCSK9 (with AAV injection). After 28days, punches werehandled and analyzed. Histology and molecular screening were performed.
As compared to WT, KO PCSK9 mice presented a 25% to 50% reduction in cholesterol, and mice overexpressing hepatic PCSK9 a 2-time higher circulating cholesterol over the course of the protocol (all P<0.003). PCSK9 circulating level was not detectable in KO and was significantly increased in mice overexpressing hepatic PCSK9 (fold change>10 vs. WT; all P<0.001). Preliminary histological analysis of explanted punches after 28days revealed an important infiltration of mononuclear cells into the pericardial matrix, consistent over the 3 groups of mice. However, the quantity and degree of infiltration of these cells appeared to be exacerbated in mice overexpressing hepatic PCSK9. Further molecular screening is ongoing.
We successfully developed an animal model allowing us the investigation of PCSK9 and hypercholesterolemia roles in SVD. Preliminary analyses suggest that high PCSK9 and/or cholesterol can potentiate cell infiltration post-implantation of bovine pericardial matrix. Further molecular analyses will provide key elements to decipher the mechanisms related to PCSK9 and cholesterol in the development of SVD.
Mitral Valve Dystrophy: What role do leukocytes play? Delwarde, Constance; Le Vély, Benjamin; Kayvanjoo, Amir H. ...
Archives of Cardiovascular Diseases Supplements,
June 2022, 2022-06-00, Letnik:
14, Številka:
2
Journal Article
Recenzirano
Odprti dostop
Mitral Valve Dystrophy (MVD) is an outgrowing disease, however little is known regarding the involved pathophysiological mechanisms. We have generated a unique knock-in (KI) animal model for the ...P637Q mutation on the FLNA gene, the first causal gene associated with MVD identified by our team. We established the relevance of this model to study MVD by confirming the presence of the disease in 3-week-old (D21) KI rats. The molecular phenotyping at this time highlighted specific biological processes such as immune cell recruitment, extracellular matrix remodeling or response to molecular stress, as central players in the development of MVD.
The aim of the present study was to analyze earlier time point (7-day post-natal; D7) and the specific role of endothelial, interstitial and immune cells in the onset and the progression of MVD.
D7 and D21 WT and KI rats were studied. Classical histology was performed at D7 to evaluate the valve remodeling. Cytometry was performed to determine the nature and the proportion of cell subpopulation at each time-points. Following cell sorting, qPCR experiments allowed the characterization of the molecular signature for each subpopulation.
The MV phenotyping at D7 confirmed the presence of MVD, but no difference was observed in myeloid cell proportion at this age (6% vs 6% for WT and KI animals, respectively; P=0.63). However, concordant with the RNA-seq data at D21, cytometry experiments revealed a 2-fold increase in the proportion of myeloid cells in MV from KI animals compared to WT (13% vs 7% respectively; P<0.05). CD45+ leukocytes and CD206+ developmental macrophages were located in the medial third and atrial border of the MV leaflet in WT rats, and more diffusely observed in KI animals at D21. Transcript levels of typical inflammatory markers (Esm1+4.3x in KI VECs vs WT, n=4; P=0.05), ECM markers (Cspg4+1.90x in KI VICs vs WT, n=5; P=0.008), and cellular activation confirmed the pro-inflammatory environment and the activation of interstitial cells in KI animals.
Our results revealed a specific role of myeloid cells in the progression of MVD rather than the onset of the disease. Furthermore, preliminary results on the cell-specific activity shows an activation of myeloid cells, endothelial and interstitial cells in MVD. Cellular cross-talk needs to be further studied to decipher the molecular mechanisms leading to MVD.
Dendritic cell-derived exosomes (Dex) are small extracellular vesicles secreted by viable dendritic cells. In the two phase-I trials that we conducted using the first generation of Dex (IFN-γ-free) ...in end-stage cancer, we reported that Dex exerted natural killer (NK) cell effector functions in patients. A second generation of Dex (IFN-γ-Dex) was manufactured with the aim of boosting NK and T cell immune responses. We carried out a phase II clinical trial testing the clinical benefit of IFN-γ-Dex loaded with MHC class I- and class II-restricted cancer antigens as maintenance immunotherapy after induction chemotherapy in patients bearing inoperable non-small cell lung cancer (NSCLC) without tumor progression. The primary endpoint was to observe at least 50% of patients with progression-free survival (PFS) at 4 mo after chemotherapy cessation. Twenty-two patients received IFN-γ-Dex. One patient exhibited a grade three hepatotoxicity. The median time to progression was 2.2 mo and median overall survival (OS) was 15 mo. Seven patients (32%) experienced stabilization of >4 mo. The primary endpoint was not reached. An increase in NKp30-dependent NK cell functions were evidenced in a fraction of these NSCLC patients presenting with defective NKp30 expression. Importantly, MHC class II expression levels of the final IFN-γ-Dex product correlated with expression levels of the NKp30 ligand BAG6 on Dex, and with NKp30-dependent NK functions, the latter being associated with longer progression-free survival. This phase II trial confirmed the capacity of Dex to boost the NK cell arm of antitumor immunity in patients with advanced NSCLC.
PDF
