Collagen 18A1/Endostatin Expression in the Progression of Right Ventricular Remodeling and Dysfunction in Pulmonary Arterial Hypertension.

Numerous studies have demonstrated that endostatin (ES), a potent angiostatic peptide derived from collagen type XVIII alpha 1 chain and encoded by COL18A1, is elevated in pulmonary arterial hypertension (PAH). Importantly, elevated ES has consistently been associated with altered hemodynamics, poor functional status, and adverse outcomes in adult and pediatric PAH. This study used serum samples from patients with Group I PAH and plasma and tissue samples derived from the Sugen/Chronic hypoxic (SuHx) rat pulmonary hypertension (PH) model to define associations between COL18A1/ES and disease development, including hemodynamics, right ventricular (RV) remodeling, and RV dysfunction. Using cardiac magnetic resonance (CMR) imaging and advanced hemodynamic assessments with pressure-volume (PV) loops in patients with PAH to assess RV-pulmonary arterial (PA) coupling, we observed a strong relationship between circulating ES levels and metrics of RV structure and function. Specifically, RV mass and the ventricular mass index (VMI) were positively associated with ES while RV ejection fraction and RV-PA coupling were inversely associated with ES levels. Our animal data demonstrates that the development of PH is associated with increased COL18A1/ES in the heart as well as the lungs. Disease-associated increases in COL18A1 mRNA and protein were most pronounced in the RV compared to the left ventricle (LV) and lung. COL18A1 expression in the RV was strongly associated with disease-associated changes in RV mass, fibrosis, and myocardial capillary density. These findings indicate that COL18A1/ES increase early in disease development in the RV and implicate COL18A1/ES in pathologic RV dysfunction in PAH.

Functional Impact of Human Genetic Variants of COL18A1/Endostatin on Pulmonary Endothelium.

Pulmonary arterial hypertension (PAH) is an incurable disease characterized by disordered and dysfunctional angiogenesis leading to small-vessel loss and an obliterative vasculopathy. The pathogenesis of PAH is not fully understood, but multiple studies have demonstrated links between elevated angiostatic factors, disease severity, and adverse clinical outcomes. ES (endostatin), one such circulating angiostatic peptide, is the cleavage product of the proteoglycan COL18A1 (collagen α1[XVIII] chain). Elevated serum ES is associated with increased mortality and disease severity in PAH. A nonsynonymous variant of ES (aspartic acid-to-asparagine substitution at amino acid 104; p.D104N) is associated with differences in PAH survival. Although COL18A1/ES expression is markedly increased in remodeled pulmonary vessels in PAH, the impact of ES on pulmonary endothelial cell (PEC) biology and molecular contributions to PAH severity remain undetermined. In the present study, we characterized the effects of exogenous ES on human PEC biology and signaling. We demonstrated that ES inhibits PEC migration, proliferation, and cell survival, with significant differences between human variants, indicating that they are functional genetic variants. ES promotes proteasome-mediated degradation of the transcriptional repressor ID1, increasing expression and release of TSP-1 (thrombospondin 1). ES inhibits PEC migration via an ID1/TSP-1/CD36-dependent pathway, in contrast to proliferation and apoptosis, which require both CD36 and CD47. Collectively, the data implicate ES as a novel negative regulator of ID1 and an upstream propagator of an angiostatic signal cascade converging on CD36 and CD47, providing insight into the cellular and molecular effects of a functional genetic variant linked to altered outcomes in PAH.

Cryopreserved human precision-cut lung slices provide an immune competent pulmonary test system for "on-demand" use and long-term cultures.

Human precision-cut lung slices (hPCLS), considered a highly relevant ex vivo model of the lung, offer native architecture and cells of the lung tissue including respiratory parenchyma, small airways, and immune competent cells. However, the irregular availability of donor lungs has limited the accessibility of this system. As described here, thousands of hPCLS can be created from 1 lung, cryopreserved, and used "on demand" by applying slicing and cryopreservation methodology improvements. Fresh and cryopreserved (∼7 and ∼34 weeks; F&C) hPCLS from 1 donor lung were cultured for up to 29 days and evaluated for biomass, viability, tissue integrity, and inflammatory markers in response to lipopolysaccharide (LPS; 5 µg/ml) and Triton X-100 (TX100; 0.1%) challenge (24 h) at days 1, 8, 15, 22, and 29 following culture initiation. The F&C hPCLS retained biomass, viability, and tissue integrity throughout the 29 days and demonstrated immune responsiveness with up to ∼30-fold LPS-induced cytokine increases. Histologically, more than 70% of normal cytomorphological features were preserved in all groups through day 29. Similar retention of tissue viability and immune responsiveness post cryopreservation (4-6 weeks) and culture (up to 14 days) was observed in hPCLS from additional 3 donor lungs. Banking cryopreserved hPCLS from various donors (and disease states) provides a critical element in researching human-derived pulmonary tissue. The retention of viability and functional responsiveness (≥4 weeks) allows evaluation of long-term, complex endpoints reflecting key events in Adverse Outcome Pathways and positions hPCLS as a valuable human-relevant model for use in regulatory applications.

Oxidation of Methionine 77 in Calmodulin Alters Mouse Growth and Behavior.

Methionine 77 in calmodulin can be stereospecifically oxidized to methionine sulfoxide by mammalian methionine sulfoxide reductase A. Whether this has in vivo significance is unknown. We therefore created a mutant mouse in which wild type calmodulin-1 was replaced by a calmodulin containing a mimic of methionine sulfoxide at residue 77. Total calmodulin levels were unchanged in the homozygous M77Q mutant, which is viable and fertile. No differences were observed on learning tests, including the Morris water maze and associative learning. Cardiac stress test results were also the same for mutant and wild type mice. However, young male and female mice were 20% smaller than wild type mice, although food intake was normal for their weight. Young M77Q mice were notably more active and exploratory than wild type mice. This behavior difference was objectively documented on the treadmill and open field tests. The mutant mice ran 20% longer on the treadmill than controls and in the open field test, the mutant mice explored more than controls and exhibited reduced anxiety. These phenotypic differences bore a similarity to those observed in mice lacking calcium/calmodulin kinase IIα (CaMKIIα). We then showed that MetO77 calmodulin was less effective in activating CaMKIIα than wild type calmodulin. Thus, characterization of the phenotype of a mouse expressing a constitutively active mimic of calmodulin led to the identification of the first calmodulin target that can be differentially regulated by the oxidation state of Met77. We conclude that reversible oxidation of methionine 77 in calmodulin by MSRA has the potential to regulate cellular function.

Antioxidant polyphenol-rich extracts from the medicinal plants Antirhea borbonica, Doratoxylon apetalum and Gouania mauritiana protect 3T3-L1 preadipocytes against H2O2, TNFα and LPS inflammatory mediators by regulating the expression of superoxide dismutase and NF-κB genes.

BACKGROUND: Adipose cells responsible for fat storage are the targets of reactive oxygen species (ROS) like H2O2 and pro-inflammatory agents including TNFα and LPS. Such mediators contribute to oxidative stress and alter inflammatory processes in adipose tissue, leading to insulin resistance during obesity. Thus, the identification of natural compounds such as plant polyphenols able to increase the antioxidant and anti-inflammatory capacity of the body is of high interest. We aimed to evaluate the biological properties of polyphenol-rich extracts from the medicinal plants A. borbonica, D. apetalum and G. mauritiana on preadipocytes exposed to H2O2, TNFα or LPS mediators. METHODS: Medicinal plant extracts were analysed for their polyphenol contents by Folin-Ciocalteu and UPLC-ESI-MS methods as well as for their free radical-scavenging activities by DPPH and ORAC assays. To assess the ability of polyphenol-rich extracts to protect 3T3-L1 preadipocytes against H2O2, TNFα or LPS mediators, several parameters including cell viability (MTT and LDH assays), ROS production (DCFH-DA test), IL-6 and MCP-1 secretion (ELISA) were evaluated. Moreover, the expression of superoxide dismutase, catalase and NF-κB genes was explored (RT-QPCR). RESULTS: All medicinal plants exhibited high levels of polyphenols with free radical-scavenging capacities. Flavonoids such as quercetin, kaempferol, epicatechin and procyanidins, and phenolic acids derived from caffeic acid including chlorogenic acid, were detected. Polyphenol-rich plant extracts did not exert a cytotoxic effect on preadipocytes but protected them against H2O2 anti-proliferative action. Importantly, they down-regulated ROS production and the secretion of IL-6 and MCP-1 pro-inflammatory markers induced by H2O2, TNFα and LPS mediators. Such a protective action was associated with an increase in superoxide dismutase antioxidant enzyme gene expression and a decrease in mRNA levels of NF-κB pro-inflammatory transcription factor. CONCLUSION: This study highlights that antioxidant strategies based on polyphenols derived from medicinal plants tested could contribute to regulate adipose tissue redox status and immune process, and thus participate to the improvement of obesity-related oxidative stress and inflammation.

Soluble HMGB1 is a novel adipokine stimulating IL-6 secretion through RAGE receptor in SW872 preadipocyte cell line: contribution to chronic inflammation in fat tissue.

Low-grade inflammation (LGI) is a central phenomenon in the genesis of obesity and insulin-resistance characterized by IL-6 in human serum. Whereas this LGI was initially thought to be mainly attributed to macrophage activation, it is now known that pre-adipocytes and adipocytes secrete several adipokines including IL-6 and participate to LGI and associated pathologies. In macrophages, HMGB1 is a nuclear yet secreted protein and acts as a cytokine to drive the production of inflammatory molecules through RAGE and TLR2/4. In this paper we tested the secretion of HMGB1 and the auto- and paracrine contribution to fat inflammation using the human preadipocyte cell line SW872 as a model. We showed that 1) human SW872 secreted actively HMGB1, 2) IL-6 production was positively linked to high levels of secreted HMGB1, 3) recombinant HMGB1 boosted IL-6 expression and this effect was mediated by the receptor RAGE and did not involve TLR2 or TLR4. These results suggest that HMGB1 is a major adipokine contributing to LGI implementation and maintenance, and can be considered as a target to develop news therapeutics in LGI associated pathologies such as obesity and type II diabetes.