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.

Molecular docking of lutein as anti-photoaging agent in silico

The accumulation of UV exposure resulted in the loss of skin elasticity, and the appearance of wrinkles on the skin is commonly known as photoaging. Matrix metalloproteinase-1 (MMP-1) is an enzyme that degrades type I and III fibrillar collagen. This study aims to determine the mechanism of MMP-1 inhibition by lutein, a carotenoid compound with high antioxidant activity, using in silico molecular docking. This study was conducted by optimization of lutein structure using HyperChem 8, preparation of MMP-1 (PDB ID: 966C) using Chimera 1.10.1, validation of the method, and docking lutein against MMP-1 using Autodock 4.2. The results showed lutein had binding energy of -12.28 kcal/mol, lower than RS2 native ligand (-10.83 kcal/mol). The hydrogen bond formed between lutein and MMP-1 through HIS228 residue. To conclude, lutein may be developed as an anti-photoaging agent by inhibiting the MMP-1.

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.

Melt electro-written scaffolds with box-architecture support orthogonally-oriented collagen

Melt Electro-Writing (MEW) is a state-of-the-art technique that supports fabrication of 3D, precisely controlled and reproducible fiber structures. A standard MEW scaffold design is a box-structure, where a repeat layer of 90° boxes is produced from a single fiber. In 3D form (i.e., multiple layers), this structure has the potential to mimic orthogonal arrangements of collagen, as observed in the corneal stroma. In this study, we determined the response of human primary corneal stromal cells and their deposited fibrillar collagen (detected using a CNA35 probe) following 6-weeks in vitro culture on these box-structures made from poly(ε-caprolactone). Comparison was also made to glass substrates (topography-free) and electrospun poly(ε-caprolactone) fibers (aligned topography). Cell orientation and collagen deposition were non-uniform on glass substrates. Electrospun scaffolds supported an excellent parallel arrangement of cells and deposited collagen to the underlying architecture of aligned fibers, but there was no evidence of bidirectional collagen. In contrast, MEW scaffolds encouraged the formation of a dense, interconnected cellular network and deposited fibrillar collagen layers with a distinct orthogonal-arrangement. Collagen fibrils were particularly dominant through the middle layers of the MEW scaffolds’ total thickness and closer examination revealed these fibrils to be concentrated within the pores’ central regions. With the demand for donor corneas far exceeding the supply - leaving many with visual impairment - the application of MEW as a potential technique to recreate the corneal stroma with spontaneous, bidirectional collagen organization warrants further study.

Widespread distribution of collagens and collagen-associated domains in eukaryotes

The origin of collagen, the dominant structural component of metazoan extracellular matrix, has long been cited as a critical step in the evolution of metazoan multicellularity. While collagens were once thought to be found only in metazoans, scattered reports of collagen domains in Fungi, and more recently in close relatives of metazoans, have called into question whether collagens are truly unique to metazoans. Here, we take advantage of recently sequenced genomes and transcriptomes of diverse holozoans (the clade encompassing metazoans and their close relatives), as well as publicly available proteomes from diverse non-holozoan eukaryotes, to conduct a systematic search for collagen domains across eukaryotic diversity. We find that collagen domains are ubiquitous in choanoflagellates, the sister group of metazoans, and widespread across many other major eukaryotic taxa. Many predicted collagens in non-metazoans are comparable to metazoan collagens in length and proline content. Moreover, most are present in species that also encode putative prolyl 4-hydroxylase domains, suggesting that, like metazoan collagens, they may be stabilized through the hydroxylation of prolines. Fibrillar collagen and collagen IV appear to be unique to metazoans, and we posit that their ability to assemble into superstructures may have contributed to the origin of metazoan multicellularity.

Real-time polarization microscopy of fibrillar collagen in histopathology

Over the past two decades, fibrillar collagen reorganization parameters such as the amount of collagen deposition, fiber angle and alignment have been widely explored in numerous studies. These parameters are now widely accepted as stromal biomarkers and linked to disease progression and survival time in several cancer types. Despite all these advances, there has not been a significant effort to make it possible for clinicians to explore these biomarkers without adding steps to the clinical workflow or by requiring high-cost imaging systems. In this paper, we evaluate previously described polychromatic polarization microscope (PPM) to visualize collagen fibers with an optically generated color representation of fiber orientation and alignment when inspecting the sample by a regular microscope with minor modifications. This system does not require stained slides, but is compatible with histological stains such as H&E. Consequently, it can be easily accommodated as part of regular pathology review of tissue slides, while providing clinically useful insight into stromal composition.

Authentication of a novel antibody to zebrafish collagen type XI alpha 1 chain (Col11a1a)

Objective Extracellular matrix proteins play important roles in embryonic development and antibodies that specifically detect these proteins are essential to understanding their function. The zebrafish embryo is a popular model for vertebrate development but suffers from a dearth of authenticated antibody reagents for research. Here, we describe a novel antibody designed to detect the minor fibrillar collagen chain Col11a1a in zebrafish (AB strain). Results The Col11a1a antibody was raised in rabbit against a peptide comprising a unique sequence within the zebrafish Col11a1a gene product. The antibody was affinity-purified and characterized by ELISA. The antibody is effective for immunoblot and immunohistochemistry applications. Protein bands identified by immunoblot were confirmed by mass spectrometry and sensitivity to collagenase. Col11a1a knockout zebrafish were used to confirm specificity of the antibody. The Col11a1a antibody labeled cartilaginous structures within the developing jaw, consistent with previously characterized Col11a1 antibodies in other species. Col11a1a within formalin-fixed paraffin-embedded zebrafish were recognized by the antibody. The antibodies and the approaches described here will help to address the lack of well-defined antibody reagents in zebrafish research.

In utero exposure to electronic-cigarette aerosols decreases lung fibrillar collagen content, increases Newtonian resistance and induces sex-specific molecular signatures in neonatal mice

Approximately 7% of pregnant women in the United States use electronic-cigarette (e-cig) devices during pregnancy. There is, however, no scientific evidence to support e-cig use as being ‘safe’ during pregnancy. Little is known about the effects of fetal exposures to e-cig aerosols on lung alveologenesis. In the present study, we tested the hypothesis that in utero exposure to e-cig aerosol impairs lung alveologenesis and pulmonary function in neonates. Pregnant BALB/c mice were exposed 2 h a day for 20 consecutive days during gestation to either filtered air or cinnamon-flavored e-cig aerosol (36 mg/mL of nicotine). Lung tissue was collected in offspring during lung alveologenesis on postnatal day (PND) 5 and PND11. Lung function was measured at PND11. Exposure to e-cig aerosol in utero led to a significant decrease in body weights at birth which was sustained through PND5. At PND5, in utero e-cig exposures dysregulated genes related to Wnt signaling and epigenetic modifications in both females (~ 120 genes) and males (40 genes). These alterations were accompanied by reduced lung fibrillar collagen content at PND5—a time point when collagen content is close to its peak to support alveoli formation. In utero exposure to e-cig aerosol also increased the Newtonian resistance of offspring at PND11, suggesting a narrowing of the conducting airways. At PND11, in females, transcriptomic dysregulation associated with epigenetic alterations was sustained (17 genes), while WNT signaling dysregulation was largely resolved (10 genes). In males, at PND11, the expression of only 4 genes associated with epigenetics was dysregulated, while 16 Wnt related-genes were altered. These data demonstrate that in utero exposures to cinnamon-flavored e-cig aerosols alter lung structure and function and induce sex-specific molecular signatures during lung alveologenesis in neonatal mice. This may reflect epigenetic programming affecting lung disease development later in life.

Differences in MMP-2 Expression in High- and Low-Grade MPNST and its Correlation with Prognostic Factors

Malignant peripheral nerve sheath tumor (MPNST) is a soft tissue sarcoma, which is difficult to distinguish from other spindle cell sarcomas. MPNST is hostile, with a high recurrence, and tends to metastasize hematogenously, especially to the lungs. A phase of the metastasis is a degradation of the extracellular matrix, where Matrix Metalloproteinase (MMP) plays an essential role in this process. Gelatinase-type MMP, MMP-2 and MMP-9, can degrade basal membrane and fibrillar collagen to open the invasion pathway. MMP-2 can degrade more collagen and non-collagen extracellular matrix than MMP-9. Therefore, the study aimed to see the relationship between MMP-2 overexpression and histopathological malignancy grading and other clinical prognostic variables. The study was conducted by immunohistochemical staining of MMP-2 in 39 cases, consisting of 19 cases of low-grade MPNST and 20 cases of high-grade MPNST. Subsequently, an analysis of the relationship between MMP-2 overexpression and the malignancy grading and clinical variables was performed, such as age, sex, and tumor size and location. MMP-2 overexpression was seen in 19 (95%) cases of high grade and three (15.8%) cases of low-grade MPNST (p 0.000). The study also found a significant relationship between MMP-2 overexpression and histopathology grading, which may be helpful to define the prognosis.