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.

Myogenic Differentiation of Human Myoblasts and Mesenchymal Stromal Stem Cells under GDF11 on Poly-ɛ-Caprolactone-Collagen I-Polyethylene-Nanofibers

Primary myoblasts (Mb) and adipose derived mesenchymal stromal cells (ADSC) can be co-cultured and myogenically differentiated in the process of skeletal muscle tissue engineering. Electrospun composite nanofiber scaffolds represent suitable matrices for tissue engineering of skeletal muscle, combining biocompatibility and stability. Although growth differentiation factor 11 (GDF11) has been proposed as a rejuvenating circulating factor, restoring skeletal muscle function in aging mice, some studies have also described a harming effect of GDF11.Therefore the aim of the study was to analyze the effect of GDF11 on co-cultures of Mb and ADSC on poly-ε-caprolacton (PCL)-collagen I-polyethylene oxide (PEO)-nanofibers.Human Mb were co-cultured with ADSC two-dimensionally (2D) as monolayers or three-dimensionally (3D) on aligned PCL-collagen I-PEO-nanofibers. Differentiation media were either serum-free with or without GDF11, or serum containing as in a conventional differentiation medium. Cell viability was higher after conventional myogenic differentiation compared to serum-free and serum-free + GDF11 differentiation as was creatine kinase activity. Immunofluorescence staining showed myosin heavy chain expression in all groups after 28 days of differentiation. Gene expression of myosin heavy chain (MYH2) increased after serum-free + GDF11 stimulation compared to serum-free stimulation alone. The results of this study show that PCL-collagen I-PEO-nanofibers represent a suitable matrix for 3D myogenic differentiation of Mb and ADSC. In this context, GDF11 seems to promote myogenic differentiation of Mb and ADSC co-cultures compared to serum-free differentiation without any evidence of a harming effect.

The Role of the Dynamic Lung Extracellular Matrix Environment on Fibroblast Morphology and Inflammation

The extracellular matrix (ECM) supports lung tissue architecture and physiology by providing mechanical stability and elastic recoil. Over the last several decades, it has become increasingly clear that the stiffness of the ECM governs many cellular processes, including cell-phenotype and functions during development, healing, and disease. Of all the lung ECM proteins, collagen-I is the most abundant and provides tensile strength. In many fibrotic lung diseases, the expression of collagen is increased which affects the stiffness of the surrounding environment. The goal of this study was to assess the effect on fibroblast morphology, cell death, and inflammation when exposed to 2D and 3D low (0.4 mg/mL) versus high (2.0 mg/mL) collagen-I-matrix environments that model the mechanics of the breathing lung. This study demonstrates that human fetal lung fibroblasts (HFL1), grown in a 3D collagen type-I environment compared to a 2D one, do not form cells with a myofibroblast morphology, express less F-actin stress fibers, exhibit less cell death, and significantly produce less pro-inflammatory IL-6 and IL-8 cytokines. Exposure to mechanical strain to mimic breathing (0.2 Hz) led to the loss of HFL1 fibroblast dendritic extensions as well as F-actin stress fibers within the cell cytoskeleton, but did not influence cytokine production or cell death. This dynamic assay gives researchers the ability to consider the assessment of the mechanodynamic nature of the lung ECM environment in disease-relevant models and the potential of mechano-pharmacology to identify therapeutic targets for treatment.

miR-15a-5p regulates myocardial fibrosis in atrial fibrillation by targeting Smad7

Background At present, there is no effective treatment for myocardial fibrosis in atrial fibrillation (AF). It is reported that miR-15a-5p is abnormally expressed in AF patients but its specific role remains unclear. This study aims to investigate the effect of miR-15a-5p in myocardial fibrosis. Methods Left atrial appendage (LAA) tissues were collected from AF and non-AF patients. In lipopolysaccharide (LPS) stimulated H9C2 cells, miR-15a-5p mimic, inhibitor, pcDNA3.1-Smad7 and small interfering RNA-Smad7 (siRNA-Smad7) were respectively transfected to up-regulate or down-regulate the intracellular expression levels of miR-15a-5p and Smad7. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot (WB) were used to determine the expression levels of miR-15a-5p, Smad7, transforming growth factor β1 (TGF-β1) and collagen I. Cell counting kit-8 (CCK-8) and ethylene deoxyuridine (EdU) were used to determine cell viability and proliferation capacity, respectively. Dual-luciferase was used to detect whether miR-15a-5p interacted with Smad7, hydroxyproline (HYP) and Hematoxylin-Eosin (HE) staining were used to detect tissue fibrosis. Results The expression levels of miR-15a-5p, TGF-β1 and collagen I were up-regulated, while Smad7 was down-regulated in AF tissues and LPS-stimulated cells. MiR-15a-5p mimic can inhibit the expression of Smad7, and the dual-luciferase experiment confirmed their interaction. MiR-15a-5p inhibitor or pcDNA3.1-Smad7 can inhibit LPS-induced fibrosis and cell proliferation, while siRNA-Smad7 can reverse the changes caused by miR-15a-5p inhibitor. Conclusion We combined clinical studies with LPS-stimulated H9C2 cell model to validate the role of miR-15a-5p in the regulation of Smad7 and fibrosis. Taken together, the miR-15a-5p/Smad7 pathway might be a potential target for AF therapy.

Stiffness Variation of 3D Collagen Networks by Surface Functionalization of Network Fibrils with Sulfonated Polymers

Fibrillar collagen is the most prominent protein in the mammalian extracellular matrix. Therefore, it is also widely used for cell culture research and clinical therapy as a biomimetic 3D scaffold. Charged biopolymers, such as sulfated glycosaminoglycans, occur in vivo in close contact with collagen fibrils, affecting many functional properties such as mechanics and binding of growth factors. For in vitro application, the functions of sulfated biopolymer decorations of fibrillar collagen materials are hardly understood. Herein, we report new results on the stiffness dependence of 3D collagen I networks by surface functionalization of the network fibrils with synthetic sulfonated polymers, namely, poly(styrene sulfonate) (PSS) and poly(vinyl sulfonate) (PVS). A non-monotonic stiffness dependence on the amount of adsorbed polymer was found for both polymers. The stiffness dependence correlated to a transition from mono- to multilayer adsorption of sulfonated polymers on the fibrils, which was most prominent for PVS. PVS mono- and multilayers caused a network stiffness change by a factor of 0.3 and 2, respectively. A charge-dependent weakening of intrafibrillar salt bridges by the adsorbed sulfonated polymers leading to fibrillar softening is discussed as the mechanism for the stiffness decrease in the monolayer regime. In contrast, multilayer adsorption can be assumed to induce interfibrillar bridging and an increase in network stiffness. Our in vitro results have a strong implication on in vivo characteristics of fibrillar collagen I, as sulfated glycosaminoglycans frequently attach to collagen fibrils in various tissues, calling for an up to now overlooked impact on matrix and tendon mechanics.

Therapeutic Effect of Seawater Pearl Powder on UV-Induced Photoaging in Mouse Skin

The objective of this study was to investigate the therapeutic effect of seawater pearl powder (SPP) on ultraviolet (UV) irradiation-induced photoaging in mouse skin. The protein and trace elements in SPP were detected by liquid chromatography-mass spectrometry, atomic fluorescence spectrometry, and inductively coupled plasma-atomic emission spectrometry. The effect of SPP on treating skin damage resulting from UV-induced photoaging was observed by gross physical appearance and histopathological analysis. Oxidative stress and melanin synthesis were analyzed using biochemical method. Western blotting was applied to analyze the phosphorylation and expression levels of matrix metalloproteinase-1 (MMP-1), collagen I, and proteins involved in the mitogen-activated protein kinase (MAPK) signaling pathways (p38, ERK, and JNK). The results show that SPP has a significant therapeutic effect on UV-induced photoaging of skin and improves and restores appearance and tissue structure of mouse skin. The major mechanism may be related to reduction of expression level of MMP-1 and enhancement of collagen I production via inhibition of MAPK signaling pathway after scavenging of excess reactive oxygen species (ROS) in the UV-induced photoaged skin of mice. Meanwhile, it may also be involved in reducing melanin content by inhibiting tyrosinase activity after scavenging excess ROS in the UV-induced photoaged skin of mice. Therefore, SPP could be a good substance to treat photoaging skin. Taking cost-effectiveness and efficacy into consideration, the optimal concentration of SPP for treating photoaging skin could be 100 mg/g.

On the Evaluation of a Novel Hypoxic 3D Pancreatic Cancer Model as a Tool for Radiotherapy Treatment Screening

Tissue engineering is evolving to mimic intricate ecosystems of tumour microenvironments (TME) to more readily map realistic in vivo niches of cancerous tissues. Such advanced cancer tissue models enable more accurate preclinical assessment of treatment strategies. Pancreatic cancer is a dangerous disease with high treatment resistance that is directly associated with a highly complex TME. More specifically, the pancreatic cancer TME includes (i) complex structure and complex extracellular matrix (ECM) protein composition; (ii) diverse cell populations (e.g., stellate cells), cancer associated fibroblasts, endothelial cells, which interact with the cancer cells and promote resistance to treatment and metastasis; (iii) accumulation of high amounts of (ECM), which leads to the creation of a fibrotic/desmoplastic reaction around the tumour; and (iv) heterogeneous environmental gradients such as hypoxia, which result from vessel collapse and stiffness increase in the fibrotic/desmoplastic area of the TME. These unique hallmarks are not effectively recapitulated in traditional preclinical research despite radiotherapeutic resistance being largely connected to them. Herein, we investigate, for the first time, the impact of in vitro hypoxia (5% O2) on the radiotherapy treatment response of pancreatic cancer cells (PANC-1) in a novel polymer (polyurethane) based highly macroporous scaffold that was surface modified with proteins (fibronectin) for ECM mimicry. More specifically, PANC-1 cells were seeded in fibronectin coated macroporous scaffolds and were cultured for four weeks in in vitro normoxia (21% O2), followed by a two day exposure to either in vitro hypoxia (5% O2) or maintenance in in vitro normoxia. Thereafter, in situ post-radiation monitoring (one day, three days, seven days post-irradiation) of the 3D cell cultures took place via quantification of (i) live/dead and apoptotic profiles and (ii) ECM (collagen-I) and HIF-1a secretion by the cancer cells. Our results showed increased post-radiation viability, reduced apoptosis, and increased collagen-I and HIF-1a secretion in in vitro hypoxia compared to normoxic cultures, revealing hypoxia-induced radioprotection. Overall, this study employed a low cost, animal free model enabling (i) the possibility of long-term in vitro hypoxic 3D cell culture for pancreatic cancer, and (ii) in vitro hypoxia associated PDAC radio-protection development. Our novel platform for radiation treatment screening can be used for long-term in vitro post-treatment observations as well as for fractionated radiotherapy treatment.

Precision 3D printed meniscus scaffolds to facilitate hMSCs proliferation and chondrogenic differentiation for tissue regeneration

Background The poor regenerative capability and structural complexity make the reconstruction of meniscus particularly challenging in clinic. 3D printing of polymer scaffolds holds the promise of precisely constructing complex tissue architecture, however the resultant scaffolds usually lack of sufficient bioactivity to effectively generate new tissue. Results Herein, 3D printing-based strategy via the cryo-printing technology was employed to fabricate customized polyurethane (PU) porous scaffolds that mimic native meniscus. In order to enhance scaffold bioactivity for human mesenchymal stem cells (hMSCs) culture, scaffold surface modification through the physical absorption of collagen I and fibronectin (FN) were investigated by cell live/dead staining and cell viability assays. The results indicated that coating with fibronectin outperformed coating with collagen I in promoting multiple-aspect stem cell functions, and fibronectin favors long-term culture required for chondrogenesis on scaffolds. In situ chondrogenic differentiation of hMSCs resulted in a time-dependent upregulation of SOX9 and extracellular matrix (ECM) assessed by qRT-PCR analysis, and enhanced deposition of collagen II and aggrecan confirmed by immunostaining and western blot analysis. Gene expression data also revealed 3D porous scaffolds coupled with surface functionalization greatly facilitated chondrogenesis of hMSCs. In addition, the subcutaneous implantation of 3D porous PU scaffolds on SD rats did not induce local inflammation and integrated well with surrounding tissues, suggesting good in vivo biocompatibility. Conclusions Overall, this study presents an approach to fabricate biocompatible meniscus constructs that not only recapitulate the architecture and mechanical property of native meniscus, but also have desired bioactivity for hMSCs culture and cartilage regeneration. The generated 3D meniscus-mimicking scaffolds incorporated with hMSCs offer great promise in tissue engineering strategies for meniscus regeneration. Graphical Abstract