Phenotypic characterization of primary cardiac fibroblasts from patients with HFpEF

Heart failure is a leading cause of hospitalizations and mortality worldwide. Heart failure with a preserved ejection fraction (HFpEF) represents a significant clinical challenge due to the lack of available treatment modalities for patients diagnosed with HFpEF. One symptom of HFpEF is impaired diastolic function that is associated with increases in left ventricular stiffness. Increases in myocardial fibrillar collagen content is one factor contributing to increases in myocardial stiffness. Cardiac fibroblasts are the primary cell type that produce fibrillar collagen in the heart. However, relatively little is known regarding phenotypic changes in cardiac fibroblasts in HFpEF myocardium. In the current study, cardiac fibroblasts were established from left ventricular epicardial biopsies obtained from patients undergoing cardiovascular interventions and divided into three categories: Referent control, hypertension without a heart failure designation (HTN (-) HFpEF), and hypertension with heart failure (HTN (+) HFpEF). Biopsies were evaluated for cardiac myocyte cross-sectional area (CSA) and collagen volume fraction. Primary fibroblast cultures were assessed for differences in proliferation and protein expression of collagen I, Membrane Type 1-Matrix Metalloproteinase (MT1-MMP), and α smooth muscle actin (αSMA). Biopsies from HTN (-) HFpEF and HTN (+) HFpEF exhibited increases in myocyte CSA over referent control although only HTN (+) HFpEF exhibited significant increases in fibrillar collagen content. No significant changes in proliferation or αSMA was detected in HTN (-) HFpEF or HTN (+) HFpEF cultures versus referent control. Significant increases in production of collagen I was detected in HF (-) HFpEF fibroblasts, whereas significant decreases in MT1-MMP levels were measured in HTN (+) HFpEF cells. We conclude that epicardial biopsies provide a viable source for primary fibroblast cultures and that phenotypic differences are demonstrated by HTN (-) HFpEF and HTN (+) HFpEF cells versus referent control.

Acazicolcept (ALPN-101), a dual ICOS/CD28 antagonist, demonstrates efficacy in systemic sclerosis preclinical mouse models

Background Uncontrolled immune response with T cell activation has a key role in the pathogenesis of systemic sclerosis (SSc), a disorder that is characterized by generalized fibrosis affecting particularly the lungs and skin. Costimulatory molecules are key players during immune activation, and recent evidence supports a role of CD28 and ICOS in the development of fibrosis. We herein investigated the efficacy of acazicolcept (ALPN-101), a dual ICOS/CD28 antagonist, in two complementary SSc-related mouse models recapitulating skin fibrosis, interstitial lung disease, and pulmonary hypertension. Methods Expression of circulating soluble ICOS and skin-expressed ICOS was investigated in SSc patients. Thereafter, acazicolcept was evaluated in the hypochlorous acid (HOCL)-induced dermal fibrosis mouse model and in the Fra-2 transgenic (Tg) mouse model. In each model, mice received 400 μg of acazicolcept or a molar-matched dose of an Fc control protein twice a week for 6 weeks. After 6 weeks, skin and lung were evaluated. Results ICOS was significantly increased in the sera from SSc patients and in SSc skin biopsies as compared to samples from healthy controls. Similar body weight changes were observed between Fc control and acazicolcept groups in both HOCL and Fra-2 Tg mice suggesting a good tolerance of acazicolcept treatment. In mice challenged with HOCL, acazicolcept induced a significant decrease in dermal thickness, collagen content, myofibroblast number, and inflammatory infiltrates characterized by B cells, T cells, neutrophils, and macrophages. In the Fra-2 Tg mouse model, acazicolcept treatment reduced lung collagen content, fibrillar collagen, histological fibrosis score, and right ventricular systolic pressure (RVSP). A reduction in frequency of CD4+ and T effector memory cells and an increase in the percentage of CD4+ T naïve cells in spleen and lung of acazicolcept-treated Fra-2 Tg mice was observed as compared to Fc control-treated Fra-2 Tg mice. Moreover, acazicolcept reduced CD69 and PD-1 expression on CD4+ T cells from the spleen and the lung. Target engagement by acazicolcept was demonstrated by blockade of CD28 and ICOS detection by flow cytometry in treated mice. Conclusions Our results confirm the importance of costimulatory molecules in inflammatory-driven fibrosis. Our data highlight a key role of ICOS and CD28 in SSc. Using complementary models, we demonstrated that dual ICOS/CD28 blockade by acazicolcept decreased dermal and pulmonary fibrosis and alleviated pulmonary hypertension. These results pave the way for subsequent research on ICOS/CD28-targeted therapies.

Effect of Proteolytic Enzymes (Bromelain and Papain) on Sensory and Chemical Quality of Sukuti (an Indigenous Dried Meat Product of Nepal)

The present work was undertaken to compare the effect of bromelain and papain on the sensory quality of sukuti (Nepalese indigenous dried buffalo meat). Buffalo lean meat (round cut) was purchased from the local market of Dharan and used for the preparation of sukuti after injection of 10% m/m enzyme (bromelain and papain) solution at the concentration (0-100 mg/L) and resting for 4h followed by drying at 65°C up to the moisture of 5%. The optimized concentration of each enzyme was selected by sensory evaluation based on color, flavor, texture, and overall acceptability. The proximate composition, collagen content, and collagen solubility were studied for two optimized samples with enzyme treatment and control. The best tenderizing effect of papain and bromelain enzyme was found at 40 mg/L and 10 mg/L respectively from the sensory analysis and the sensory attributes for these concentrations were significantly (p<0.05) higher than other samples. The soluble collagen content increased from 0.44 mg/g tissue in untreated meat to 0.52 for bromelain treated and to 0.98 mg/g for papain treated samples. The collagen solubility for the untreated sample was 4.74% which increases to 7.80% for bromelain and 13.82% for papain-treated samples. The protein content of optimized papain and bromelain treated samples was significantly decreased (p<0.05) from 82.44 to 80.25% and 81.43% respectively for papain and bromelain treated samples. There were no significant changes in fat, ash, and moisture on enzyme treatment.

Tissue Engineering of Canine Cartilage from Surgically Debrided Osteochondritis Dissecans Fragments

This study in dogs explored the feasibility of using cartilage fragments removed and discarded during routine palliative surgery for osteochondritis dissecans (OCD) as a source of primary chondrocytes for scaffold-free cartilage tissue-engineering. Primary chondrocytes were obtained from three OCD donors and one age-matched healthy articular cartilage (HAC) donor. After monolayer expansion of primary cells, a three-dimensional spherical suspension culture was implemented. Following this stage, cells were seeded at a high density into custom-made agarose molds that allowed for size and shape-specific constructs to be generated via a method of cellular self-assembling in a scaffold-free environment. Fifty-eight neocartilage constructs were tissue-engineered using this methodology. Neocartilage constructs and native cartilage from shoulder joint were subjected to histological, mechanical, and biochemical testing. OCD and HAC chondrocytes-sourced constructs had uniformly flat morphology and histology consistent with cartilage tissue. Constructs sourced from OCD chondrocytes were 1.5-times (32%) stiffer in compression and 1.3 times (23%) stronger in tension than constructs sourced from HAC chondrocytes and only 8.7-times (81%) less stiff in tension than native tissue. Constructs from both cell sources consistently had lower collagen content than native tissue (22.9%/dry weight [DW] for OCD and 4.1%/DW for HAC vs. 51.1%/DW native tissue). To improve the collagen content and mechanical properties of neocartilage, biological and mechanical stimuli, and thyroid hormone (tri-iodothyronine) were applied to the chondrocytes during the self-assembling stage in two separate studies. A 2.6-fold (62%) increase in compressive stiffness was detected with supplementation of biological stimuli alone and 5-fold (81%) increase with combined biological and mechanical stimuli at 20% strain. Application of thyroid hormone improved collagen content (1.7-times, 33%), tensile strength (1.8-times, 43%), and stiffness (1.3-times, 21%) of constructs, relative to untreated controls. Collectively, these data suggest that OCD chondrocytes can serve as a reliable cell source for cartilage tissue-engineering and that canine chondrocytes respond favorably to biological and mechanical stimuli that have been shown effective in chondrocytes from other animal species, including humans.

The Stiffness of Cardiac Fibroblast Substrates Exerts a Regulatory Influence on Collagen Metabolism via α2β1 Integrin, FAK and Src Kinases

Information about mechanical strain in the extracellular space is conducted along collagen fibers connected with integrins and then transmitted within cells. An aim of the study is to verify the hypothesis that the stiffness of cardiac human fibroblast substrates exerts a regulatory effect on collagen metabolism via integrin α2β1 and downstream signaling. The experiments were performed on human cardiac fibroblasts cultured on stiff or soft polyacrylamide gels. Extracellular and intracellular collagen content, metalloproteinase-1 (MMP-1), metalloproteinase-9 (MMP-9) and expression of the α1 chain of the procollagen type I gene (Col1A1) were elevated in cultures settled on soft substrate. The substrate stiffness did not modify tissue inhibitors of matrix metalloproteinase capacity (TIMPs 1–4). Integrin α2β1 inhibition (TC-I 15) or α2 subunit silencing resulted in augmentation of collagen content within the culture. Expression of Col1A1 and Col3A1 genes was increased in TC-I 15-treated fibroblasts. Total and phosphorylated levels of both FAK and Src kinases were elevated in fibroblasts cultured on stiff substrate. Inhibition of FAK (FAK kinase inhibitor 14) or Src kinase (AZM 47527) increased collagen content within the culture. The substrate stiffness exerted a regulatory influence on collagen metabolism via integrin α2β1 and its downstream signaling (FAK and Src kinases) in cardiac fibroblasts.

Altered Expression of ORAI and STIM Isoforms in Activated Human Cardiac Fibroblasts

Cardiac fibrotization is a well-known process characteristic of many cardiac pathological conditions. The key element is excessive activation of cardiac fibroblasts, their transdifferentiation into myofibroblasts, increased production, and accumulation of extracellular matrix proteins, resulting in cardiac stiffness. The exact cellular mechanisms and molecular components involved in the process are not fully elucidated, but the SOCE mechanism could play an important role. Its key molecules are the molecular sensor of calcium in ER/SR – STIM and the highly selective calcium channels Orai located in the plasma membrane. This study aims to evaluate selected SOCE-associated genes in the activation of HCF cell culture by several known substances (phenylephrine, isoprenaline) that represent cardiovascular overload. After cell cultivation, cell medium was collected to measure the soluble collagen content. From the harvested cells, qRT-PCR was performed to determine the mRNA levels of the corresponding genes. The activation of cells was based on changes in the relative expression of collagen genes as well as the collagen content in the medium of the cell culture. We detected an increase in the expression of the Orai2 isoform, a change in the Orai1/Orai3 ratio and also an increase in the expression of the STIM2 isoform. These results suggest an increased activation of the SOCE mechanism under stress conditions of fibroblasts, which supports the hypothesis of fibroblast activation in pathological processes by altering calcium homeostasis through the SOCE mechanism.

A neutralizing IL-11 antibody reduces vessel hyperplasia in a mouse carotid artery wire injury model

Vascular restenosis remains a major problem in patients with coronary artery disease (CAD) and peripheral artery disease (PAD). Neointimal hyperplasia, defined by post-procedure proliferation and migration of vascular smooth muscle cells (VSMCs) is a key underlying pathology. Here we investigated the role of Interleukin 11 (IL-11) in a mouse model of injury-related plaque development. Apoe−/− mice were fed a hyperlipidaemic diet and subjected to carotid wire injury of the right carotid. Mice were injected with an anti-IL11 antibody (X203), IgG control antibody or buffer. We performed ultrasound analysis to assess vessel wall thickness and blood velocity. Using histology and immunofluorescence approaches, we determined the effects of IL-11 inhibition on VSMC and macrophages phenotypes and fibrosis. Treatment of mice with carotid wire injury using X203 significantly reduced post-endothelial injury vessel wall thickness, and injury-related plaque, when compared to control. Immunofluorescence staining of the injury-related plaque showed that X203 treatment did not reduce macrophage numbers, but reduced the number of VSMCs and lowered matrix metalloproteinase 2 (MMP2) levels and collagen content in comparison to control. X203 treatment was associated with a significant increase in smooth muscle protein 22α (SM22α) positive cells in injury-related plaque compared to control, suggesting preservation of the contractile VSMC phenotype. Interestingly, X203 also reduced the collagen content of uninjured carotid arteries as compared to IgG, showing an additional effect on hyperlipidemia-induced arterial remodeling in the absence of mechanical injury. Therapeutic inhibition of IL-11 reduced vessel wall thickness, attenuated neointimal hyperplasia, and has favorable effects on vascular remodeling following wire-induced endothelial injury. This suggests IL-11 inhibition as a potential novel therapeutic approach to reduce arterial stenosis following revascularization in CAD and PAD patients.

Elaboration of pâté using fish residues

Knowing the potential of fish waste for the preparation of pâtés, there is the possibility of adding greater sustainability to the aquaculture sector. The aim was to prepare pâtés from fish processing residues with the inclusion of smoked fishmeal and evaluate their sensory, microbiological, physicochemical, and shelf-life characteristics. Three treatments were used: pâté without fishmeal inclusion (PSF), with smoked salmon carcass meal inclusion (PFSD), and smoked tilapia carcass meal (PFTD). The inclusion of the flours reduced the moisture, carbohydrate, and water activity. However, they increased the ash, salt, and collagen content. The lipid content was higher for the PFSD. The PSF showed peak oxidation at 15 days and the PFSD and PFTD around 45 days over the course of the 90-day shelf-life. The luminosity and b* coordinate were lower for PFTD, while the a* coordinate was higher for PFSD. Only the b* coordinate showed changes in PSF and PFSD throughout the 30 days of shelf-life, and its color tended to yellow at 15 days. The sensory analysis did not differ statistically between treatments (p > 0.05) for all attributes, except color, where the highest score was attributed to PSF. As for the overall impression, PSF also obtained the highest score, which may be associated with its color. By including fish flours, the nutritional composition and color of the tilapia-trimmed pâtés are changed, achieving an average acceptance level of 67%.

ALPN-101 (Acazicolcept) a Dual ICOS/CD28 Antagonist, Demonstrates Efficacy in Systemic Sclerosis Preclinical Mouse Models

BackgroundUncontrolled immune response with T cell activation has a key role in the pathogenesis of systemic sclerosis (SSc), a disorder that is characterised by generalized fibrosis affecting particularly the lungs and skin. Co-stimulatory molecules are key players during immune activation, and recent evidence supports a role of CD28 and ICOS in the development of fibrosis. We herein investigated the efficacy of ALPN-101 (acazicolcept), a dual ICOS/CD28 antagonist, in two complementary SSc-related mouse models recapitulating skin fibrosis, interstitial lung disease, and pulmonary hypertension. MethodsExpression of circulating soluble ICOS and skin-expressed ICOS was investigated in SSc patients. Thereafter, ALPN-101 was evaluated in the hypochlorous acid (HOCL)-induced dermal fibrosis mouse model and in the Fra-2 transgenic (Tg) mouse model. In each model, mice received 400 µg of ALPN-101 or a molar-matched dose of an Fc control protein twice a week for six weeks. After six weeks, skin and lung were evaluated.Results ICOS was significantly increased in the sera from SSc patients and in SSc skin biopsies as compared to samples from healthy controls. Similar body weight changes were observed between Fc Control and ALPN-101 groups in both HOCL and Fra-2 Tg mice suggesting a good tolerance of ALPN-101 treatment. In mice challenged with HOCL, ALPN-101 induced a significant decrease in dermal thickness, collagen content, myofibroblast number and inflammatory infiltrates characterized by B cells, T cells, neutrophils, and macrophages. In the Fra-2 Tg mouse model, ALPN-101 treatment reduced lung collagen content, fibrillar collagen, histological fibrosis score, and right ventricular systolic pressure (RVSP). A reduction in frequency of CD4+ and T effector memory cells and an increase in the percentage of CD4+ T naïve cells in spleen and lung of ALPN-101-treated Fra-2 Tg mice was observed as compared to Fc control-treated Fra-2 Tg mice. Moreover, ALPN-101 reduced CD69 and PD-1 expression on CD4+ T cells from the spleen and the lung. Target engagement by ALPN-101 was demonstrated by blockade of CD28 and ICOS detection by flow cytometry in treated mice. Conclusions Our results confirm the importance of co-stimulatory molecules in inflammatory-driven fibrosis. Our data highlight a key role of ICOS and CD28 in SSc. Using complementary models, we demonstrated that dual ICOS/CD28 blockade by ALPN-101 decreased dermal and pulmonary fibrosis and alleviated pulmonary hypertension. These results pave the way for subsequent research on ICOS/CD28-targeted therapies.