Levels Of Extracellular Matrix Proteins Research Articles

Identification of extracellular matrix proteins in plasma as a potential biomarker for intervertebral disc degeneration.

Recently, there has been significant focus on extracellular matrix proteolysis due to its importance in the pathological progression of intervertebral disc degeneration (IVDD). The present study investigates the circulating levels of extracellular matrix proteins in the plasma of IVDD and determines their potential relevance as biomarkers in disc degeneration. Global proteomic analysis was performed in the plasma samples of 10 healthy volunteers (HV) and 10 diseased subjects (DS) after depletion of highly abundant proteins such as albumin and IgG. We identified 144 and 135 matrix-associated proteins in plasma samples from healthy volunteers (HV) and patients with disc degeneration (DS), respectively. Among these, 49 of the matrix-associated proteins were identical to the proteins found in intervertebral disc (IVD) tissues retrieved from the in-house library. Applying stringent parameters, we selected 28 proteins, with 26 present in DS and 21 in HV. 19 proteins were found common between the groups, two of which-aggrecan (ACAN) and fibulin 1 (FBLN1) - showed statistically significant differences. Specifically, ACAN was up-regulated and FBLN1 was down-regulated in the DS-plasma. In particular, DS-plasma exhibited specific expression of collagen type 2a1 (COL2A1), native to the nucleus pulposus. The distinct presence of collagen type 2a1 and the elevated expression of aggrecan in IVDD plasma may serve as the basis for the development of a potential biomarker for monitoring the progression of disc degeneration.

Increased levels of extracellular matrix proteins associated with extracellular vesicles from brains of aged mice.

Extracellular vesicles (EVs) are secreted by all major cell types of the brain, providing a mode of intercellular communication and a pathway for disposal of cellular debris. EVs help maintain healthy brain function, but may also contribute to diseases affecting the brain. EVs might contribute to aging of the brain, as aging-related processes such as inflammation and cellular senescence may alter EV cargo, promoting further inflammation and senescence. However, the effects of aging on brain EVs and the function of EVs in the aging brain remain poorly understood. To address this question, we measured the levels and protein cargo of EVs isolated from the brains of 4-, 12-, and 22-month-old C57BL/6J mice. We detected no changes in EV levels, but observed age-dependent changes in EV proteins. EV fractions from aged (22 month old) brains contained higher levels of extracellular matrix proteins than EV fractions from young (4 month old) brains, with intermediate levels in 12-month-old brains. Specifically, EV fractions from aged mice contained elevated levels of hyaluronan and proteoglycan link proteins 1 and 2 and several chondroitin sulfate proteoglycans (CSPGs). Analysis of extracellular matrix in several brain regions of aged mice revealed increased immunolabeling for the CSPG aggrecan, but reduced labeling with Wisteria floribunda agglutinin, which binds to chondroitin sulfate side chains of CSPGs. These data are consistent with prior studies showing changes to the composition of extracellular matrix in aged brains, and indicate a novel association of EVs with changes in the extracellular matrix of the aging brain.

Silencing LncRNA SNHG14 alleviates renal tubular injury via the miR-483-5p/HDAC4 axis in diabetic kidney disease.

This study explored the clinical value of long non-coding RNA small nucleolar RNA host gene 14 (SNHG14) in diabetic kidney disease (DKD) and the mechanism of renal tubular injury. Patients with DKD, type 2 diabetes mellitus (T2DM) and healthy individuals (HVs) were included, as well as the human proximal tubular epithelial cell line (HK-2) induced by high glucose was also included. The mRNA levels of SNHG14 in the serum and cells were detected using RT-qPCR. Diagnostic significance was examined using receiver operating characteristic (ROC) analysis. A commercial test kit, flow cytometry, and enzyme-linked immunosorbent assays were employed to assess reactive oxygen species (ROS) production, apoptosis, inflammatory factor secretion, and extracellular matrix protein levels in HK-2 cells. The dual-luciferase reporter assay and RNA immunoprecipitation were used to validate miR-483-5p concerning SNHG14 or histone deacetylase 4 (HDAC4). SNHG14 and HDAC4 levels were elevated in the serum of DKD patients and HG-induced HK-2 cells, while miR-483-5p levels were decreased (P < 0.001). SNHG14 increased HDAC4 levels by sponging miR-483-5p. Elevated SNHG14 levels significantly differentiated DKD patients from HVs (AUC = 0.944) and T2DM (AUC = 0.867). Silencing of SNHG14 alleviated HG-induced ROS production and apoptosis as well as the over-secretion of inflammatory factors and extracellular matrix proteins; however, this alleviation was typically suppressed by low expression of miR-483-5p (P < 0.001). Elevated miR-483-5p alleviates HG-induced renal tubular injury, but this alleviation is suppressed by HDAC4 overexpression. In summary, suppression of SNHG14 has been shown in our study to mitigate renal tubular injury in DKD by regulating apoptosis, oxidative stress, inflammation, and fibrosis through the miR-483-5p/HDAC4 axis.

Identification of a fibronectin-binding protein signature associated with idiopathic pulmonary fibrosis

The extracellular matrix (ECM) is a critical component of tissue where it provides structural and signaling support to cells. Its dysregulation and accumulation lead to fibrosis, a major clinical challenge underlying many diseases that currently has little effective treatment. An understanding of the key molecular initiators of fibrosis would be both diagnostically useful and provide potential targets for therapeutics. The ECM protein fibronectin (FN) is upregulated in fibrotic conditions and other ECM proteins depend on assembly of a FN foundational ECM for their matrix incorporation. We used cell culture and in vivo models to investigate the role of FN in the progression of lung fibrosis. We confirmed that normal human lung fibroblasts (NHLFs) treated with transforming growth factor-beta (TGF-β) to stimulate fibrotic gene expression significantly increased both FN expression and its assembly into a matrix. We found that levels of alternatively spliced EDA and EDB exons were proportional to the increase in total FN RNA and protein showing that inclusion of these exons is not enhanced by TGF-β stimulation. RNA-sequencing identified 43 core matrisome genes that were significantly up- or down-regulated by TGF-β treatment and a Luminex immunoassay demonstrated increased levels of ECM proteins in conditioned medium of TGF-β-treated NHLFs. Interestingly, among the regulated core matrisome genes, 16 encode known FN-binding proteins and, of these, insulin-like growth factor binding protein 3 (IGFBP3) was most highly up-regulated. To link the NHLF results with in vivo disease, we analyzed lung tissue and bronchoalveolar lavage fluid from bleomycin-treated mice and found dramatically higher levels of FN and the FN-binding proteins IGFBP3, tenascin-C, and type I collagen in fibrotic conditions compared to controls. Altogether, our data identify a set of FN-binding proteins whose upregulation is characteristic of IPF and suggest that FN provides the foundational matrix for deposition of these proteins as fibrosis develops.

TGFβ1-Induced Fibrotic Responses of Conjunctival Fibroblasts through the Wnt/β-Catenin/CRYAB Signaling Pathway

Conjunctival fibrosis (CF) is a common postoperative complication of glaucoma filtration surgery (GFS), resulting in uncontrolled intraocular pressure and surgery failure. Therefore, it’s urgent to understand the molecular mechanisms underlying CF and explore novel pharmacological anti-fibrosis therapies for GFS. Our 4D-DIA quantitative proteomic results, coupled with experimental data, revealed the activation of the Wnt/β-catenin pathway in TGFβ1-induced human conjunctival fibroblasts (HConFs). Treatment with ICG-001, a Wnt/β-catenin inhibitor, effectively inhibited cell proliferation and migration in TGFβ1-treated HConFs. ICG-001 treatment alleviated the increased generation of extracellular matrix (ECM) proteins induced by TGFβ1. In addition, ICG-001 reduced the expression level of alpha smooth muscle actin (α-SMA) and inhibited cell contractility in TGFβ1-treated HConFs. Proteomics data further suggested that CRYAB was a downstream target of the Wnt/β-catenin, which was upregulated by TGFβ1 and downregulated by ICG-001. Immunoblotting assay also indicated that ICG-001 reduced the expressions of ubiquitin and β-catenin in TGFβ1-treated HConFs, implying that CRYAB stabilized β-catenin by inhibiting its ubiquitination degradation. Exogenous CRYAB promoted cell viability, increased ECM proteins levels, and upregulated α-SMA expression of HConFs under TGFβ1 stimulation. Furthermore, CRYAB rescued TGFβ1-induced fibrotic responses that were suppressed by ICG-001. In conclusion, our study elucidates the regulatory mechanism of Wnt/β-catenin/CRYAB pathway in CF, offering promising therapeutic targets for mitigating bleb scarring after GFS.

Harnessing developmental dynamics of spinal cord extracellular matrix improves regenerative potential of spinal cord organoids

Neonatal spinal cord tissues exhibit remarkable regenerative capabilities as compared to adult spinal cord tissues after injury, but the role of extracellular matrix (ECM) in this process has remained elusive. Here, we found that early developmental spinal cord had higher levels of ECM proteins associated with neural development and axon growth, but fewer inhibitory proteoglycans, compared to those of adult spinal cord. Decellularized spinal cord ECM from neonatal (DNSCM) and adult (DASCM) rabbits preserved these differences. DNSCM promoted proliferation, migration, and neuronal differentiation of neural progenitor cells (NPCs) and facilitated axonal outgrowth and regeneration of spinal cord organoids more effectively than DASCM. Pleiotrophin (PTN) and Tenascin (TNC) in DNSCM were identified as contributors to these abilities. Furthermore, DNSCM demonstrated superior performance as a delivery vehicle for NPCs and organoids in spinal cord injury (SCI) models. This suggests that ECM cues from early development stages might significantly contribute to the prominent regeneration ability in spinal cord.

Abstract 3077: Integrin expression during ovarian cancer progression

Abstract During peritoneal dissemination, ovarian cancer cells exfoliate from the primary tumor and aggregate in the peritoneal fluid. This elicits survival signals via changes in mitochondrial morphology and function. As spheroids adhere to secondary sites, mitochondrial function is restored in the form of recovery of mitochondrial morphology, increased mitochondrial membrane potential, glucose uptake and increased proliferation. These findings suggested a mitochondrial phenotypic switch from mitophagy in spheroids to upregulation of mitobiogenesis upon spheroids adhesion to secondary sites. Hence, our objective was to identify critical receptors that facilitate the adhesion of spheroids to secondary sites, focusing on integrins that facilitate cell-extracellular matrix (ECM) attachment and activate downstream signaling cascades. To investigate changes in integrin expression, we mimicked three major stages of ovarian cancer progression by developing adherent stage, spheroid stage and adherent spheroid stage using our mouse ovarian surface epithelial (MOSE) cell lines representing benign (MOSE-E) stage as well as slow-developing (MOSE-L) and fast-developing disease (MOSE-LTICv) in biologically relevant conditions. Using RT qPCR and Western blotting, we determined gene and protein expression levels of integrins and adhesion related genes. Confocal microscopy was used to determine protein localization during ovarian cancer progression. RT qPCR result indicated differential up-regulated expression of integrins in both spheroid and adherent spheroid stage, suggesting the role of different integrins during the stages of ovarian cancer progression. Specifically, we observed a decrease (integrin ɑ5, ɑ6) or increase (ɑ2𝛃;1) of integrins after aggregation and an upregulation of 𝛃;1, α2, αV, α3, α5 and immunomodulatory integrins such as αE and αM in adherent spheroids, indicating their role in mediating specific steps during progression of ovarian cancer from tumorigenesis to metastatic implantation. We also observed increased expression levels of extracellular matrix proteins and their modulators, and cell-to-cell adhesion genes during ovarian cancer progression that was more pronounced in the MOSE-LTICv cells which suggests their association with an aggressive cancer phenotype. Overall, this project will aid in the identification of adhesion targets to develop new anti-metastatic treatments that will reduce mortality in women with ovarian cancer. Citation Format: Nazia Bano, Eva M. Schmelz. Integrin expression during ovarian cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3077.

Frequent Premature Atrial Contractions Lead to Adverse Atrial Remodeling and Atrial Fibrillation in a Swine Model.

Frequent premature atrial complexes (PACs) are associated with future incident atrial fibrillation (AF), but whether PACs contribute to development of AF through adverse atrial remodeling has not been studied. This study aimed to explore the effect of frequent PACs from different sites on atrial remodeling in a swine model. Forty swine underwent baseline electrophysiologic studies and echocardiography followed by pacemaker implantations and paced PACs (50% burden) at 250-ms coupling intervals for 16 weeks in 4 groups: (1) lateral left atrium (LA) PACs by the coronary sinus (Lat-PAC; n=10), (2) interatrial septal PACs (Sep-PAC; n=10), (3) regular LA pacing at 130 beats/min (Reg-130; n=10), and (4) controls without PACs (n=10). At the final study, repeat studies were performed, followed by tissue histology and molecular analyses focusing on fibrotic pathways. Lat-PACs were associated with a longer P-wave duration (93.0±9.0 versus 74.2±8.2 and 58.8±7.6 ms; P<0.001) and greater echocardiographic mechanical dyssynchrony (57.5±11.6 versus 35.7±13.0 and 24.4±11.1 ms; P<0.001) compared with Sep-PACs and controls, respectively. After 16 weeks, Lat-PACs led to slower LA conduction velocity (1.1±0.2 versus 1.3±0.2 [Sep-PAC] versus 1.3±0.1 [Reg-130] versus 1.5±0.2 [controls] m/s; P<0.001) without significant change in atrial ERP. The Lat-PAC group had a significantly increased percentage of LA fibrosis and upregulated levels of extracellular matrix proteins (lysyl oxidase and collagen 1 and 8), as well as TGF-β1 (transforming growth factor-β1) signaling proteins (latent and monomer TGF-β1 and phosphorylation/total ratio of SMAD2/3; P<0.05). The Lat-PAC group had the longest inducible AF duration (terminal to baseline: 131 [interquartile range 30, 192] seconds versus 16 [6, 26] seconds [Sep-PAC] versus 22 [11, 64] seconds [Reg-130] versus -1 [-16, 7] seconds [controls]; P<0.001). In this swine model, frequent PACs resulted in adverse atrial structural remodeling with a heightened propensity to AF. PACs originating from the lateral LA produced greater atrial remodeling and longer induced AF duration than the septal-origin PACs. These data provide evidence that frequent PACs can cause adverse atrial remodeling as well as AF, and that the location of ectopic PACs may be clinically meaningful.

Adiponectin C1q/Tumor Necrosis Factor-Related Protein 13 (CTRP13) Protects against Renal Inflammation and Fibrosis in Obstructive Nephropathy.

Renal inflammation and fibrosis are the important pathological phenomena associated with obstructive nephropathy. However, the underlying mechanism associated with this disease has yet to be fully elucidated. The present study, therefore, aimed to investigate the effects mediated by C1q/tumor necrosis factor-related protein 13 (CTRP13) on renal inflammation and fibrosis in addition to elucidating the underlying mechanism. To meet this aim, a mouse unilateral ureteral obstruction (UUO)-mediated renal dysfunction model was established. In addition, hematoxylin-eosin staining (H&E) staining and immunofluorescence experiments as well as Western blotting and reverse transcription quantitative (RT q) PCR analyses were performed. Recombinant CTRP13 was used to investigate the role of CTRP13 in chronic renal inflammation and fibrosis. A decreased expression level of CTRP13 was identified in the plasma of patients with renal fibrosis and in UUO-model mice. The renal histopathological and functional analyses revealed that CTRP13 could both reverse UUO mediated renal dysfunction and ameliorate the conditions of tubulointerstitial fibrosis and tubular injury. Additionally, CTRP13 was found to inhibit the expression levels of extracellular matrix proteins and proinflammatory mediators. In terms of the underlying mechanism, the protective effects on inflammation and fibrosis of the kidneys of CTRP13-treated mice undergoing UUO were found to be associated with the inactivation of the TGF β/Smad and NF κB p65 signaling pathways. Taken together, these findings have suggested that CTRP13 fulfills a vital role in the progression of obstructive nephropathy, thereby uncovering brand new insights into possible leads for the therapeutic treatment of chronic kidney disease (CKD).

FBN1 knockout promotes cervical artery dissection by inducing N-glycosylation alternation of extracellular matrix proteins in rat VSMCs

FBN1 mutation promotes the degeneration of microfibril structures and extracellular matrix (ECM) integrity in the tunica media of the aorta in Marfan syndrome. However, whether FBN1 modulates cervical artery dissection (CAD) development and the potential molecular mechanisms of abnormal FBN1 in CAD remains elusive. In this study, FBN1 deficiency participated in the development of CAD and influenced the proliferation, apoptosis, and migration of vascular smooth muscle cells. FBN1 knockout induced alternations in mRNA levels of the transcriptome, protein expression of the proteome, and abundance of N-glycosylation of the N-glycoproteome. Comprehensive analysis of multiple omics showed up-regulation in mRNA levels of ECM proteins; yet, both the ECM protein levels and relative abundance of N-glycosylation were decreased. Moreover, we performed in vivo experiments to confirm the altered glycosylation of proteins in vascular smooth muscle cells. In conclusion, FBN1 deletion in vascular smooth muscle cells can result in altered N-glycosylation of ECM protein, which were critical for the stability of ECM and the process of CAD. This may open the way for a novel therapeutic strategy to treat people with CAD.

Effective electrical stimulation by a Poly(l-lactic acid)/Vitamin B2-Based piezoelectric generator promotes wound healing

Chronic wounds pose an increasing burden not only to patients, but also to the health system. Wound repair requires the timely and orderly migration of fibroblasts to the wound bed. In this respect, electrical stimulation by a piezoelectric generator (PEG) is an attractive approach to wound management because it is noninvasive and effective in promoting chronic wound healing. However, commonly used PEGs based on polyvinylidenefluoride (PVDF) and poly(l-lactic acid) (PLLA) suffer from low biocompatibility and low piezoelectric output, respectively. Herein, we developed a bio-based PEG composed of PLLA blended with vitamin B2 (VB2), leading to an improvement of crystallinity and β-phase orientation, which exhibited better biocompatibility than PVDF and a higher piezoelectric output than pure PLLA. Such piezoelectricity could serve as effective electrical stimulation and promoted in vitro cell migration and fibroblast proliferation, evidenced by significant higher gene expression levels of extracellular matrix proteins and growth factors. Moreover, it facilitated in vivo wound closure by enhancing the re-epithelialization, collagen deposition, neovascularization, and increase the concentration of growth factors in the wound bed, leading to a 1.4-fold higher wound closure rate than that in the control group. Thus, the PLLA/VB2 PEG opens an avenue for the application of bio-based PEGs for wound healing.

Pre-selection of fibroblast subsets prompts prevascularization of tissue engineered skin analogues.

The papillary and reticular dermis harbors phenotypically distinct fibroblasts, whose functions such as maintenance of skin's microvasculature are also distinct. Thus, we hypothesized that pre-selection of the subpopulations of fibroblasts would benefit the generation of skin tissue engineered (TE) constructs, promoting their prevascularization in vitro. We first isolated papillary and reticular fibroblasts using fluorescence-activated cell sorting and studied the effect of their secretome and extracellular matrix (ECM) on human dermal microvascular endothelial cell (hDMEC) organization. Subsequently, we developed a bilayered 3D polymeric structure with distinct layer-associated features to house the subpopulations of fibroblasts, to generate a skin analogue. Both papillary and reticular fibroblasts were able to stimulate capillary-like network formation in a Matrigel assay. However, the secretome of the two subpopulations was substantially different, being enriched in VEGF, IGF-1, and Angio-1 in the case of papillary fibroblasts and in HGF and FGF-2 for the reticular subset. In addition, the fibroblast subpopulations deposited varied levels of ECM proteins, more collagen I and laminin was produced by the reticular subset, but these differences did not impact hDMEC organization. Vessel-like structures with lumens were observed earlier in the 3D skin analogue prepared with the sorted fibroblasts, although ECM deposition was not affected by the cell's pre-selection. Moreover, a more differentiated epidermal layer was obtained in the skin analogue formed by the sorted fibroblasts, confirming that its whole structure was not affected. Overall, we provide evidence that pre-selection of papillary and reticular fibroblasts is relevant for promoting the in vitro prevascularization of skin TE constructs.

NEDD8-Activating Enzyme Inhibitor MLN4924 Inhibits Both the Tumor Stroma and Angiogenesis in Pancreatic Cancer via Gli1 and REDD1.

Pancreatic cancer is characterized by a dense desmoplasia stroma, which hinders efficient drug delivery and plays a critical role in tumor progression and metastasis. MLN4924 is a first-in-class NEDD8-activating enzyme inhibitor that exhibits anti-tumor activities toward pancreatic cancer, and given the comprehensive effects that MLN4924 could have, we ask what impact MLN4924 would have on the stroma of pancreatic cancer and its underlying mechanisms. Primary pancreatic stellate cells (PSCs) and human HMEC-1 cells were treated with MLN4924 in vitro. The proliferation and extracellular matrix protein levels of PSCs were tested, and their relationship with transcription factor Gli1 in PSCs was investigated. The angiogenic phenotypes of HMEC-1 cells were evaluated using capillary-like tube formation assay, and their relationship with REDD1 in HMEC-1 cells was investigated. In this study, we found that MLN4924 inhibited the proliferation of pancreatic stellate cells and their secretion of collagen and CXCL-1, and the collagen secretion inhibiting effect of MLN4924 was related with transcription factor Gli1. MLN4924 inhibited multiple angiogenic phenotypes of HMEC-1 cells, and mTOR agonist partially relieved the inhibition of MLN4924 on HEMCs. MLN4924 increased the expression of REDD1 and REDD1 knockdown promoted the angiogenic phenotypes of HMEC-1 cells. Our study suggests that MLN4924 inhibits both the tumor stroma and angiogenesis in pancreatic cancer, and the inhibition effect is related with Gli1 in pancreatic stellate cells and REDD1 in vascular endothelial cells, respectively.

Characterization of a mGluR5 Knockout Rat Model with Hallmarks of Fragile X Syndrome.

The number of reported cases of neurodevelopmental disorders has increased significantly in the last few decades, but the etiology of these diseases remains poorly understood. There is evidence of a fundamental link between genetic abnormalities and symptoms of autism spectrum disorders (ASDs), and the most common monogenetic inheritable form of ASDs is Fragile X Syndrome (FXS). Previous studies indicate that FXS is linked to glutamate signaling regulation by the G-protein-coupled metabotropic glutamate receptor 5 (mGluR5), which has been shown to have a regulatory role in neuroinflammation. We characterized the effect of knocking out mGluR5 in an organism known to have complex cognitive functions—the rat. The heterozygous phenotype is the most clinically relevant; therefore, we performed analysis in heterozygous pups. We showed developmental abnormalities in heterozygous mGluR5 knockout rats, as well as a significant increase in chemokine (C-X-C motif) ligand 1 (CXCL) expression, a hallmark indicator of early onset inflammation. We quantified an increase in microglial density in the knockout pups and quantified morphological phenotypes representative of greater reactivity in the male vs. female and postnatal day 28 heterozygous pups compared to postnatal day 14 heterozygous pups. In response to injury, reactive microglia release matrix metalloproteases, contribute to extracellular matrix (ECM) breakdown, and are responsible for eradicating cellular and molecular debris. In our study, the changes in microglial density and reactivity correlated with abnormalities in the mRNA expression levels of ECM proteins and with the density of perineuronal nets. We saw atypical neuropsychiatric behavior in open field and elevated plus tests in heterozygous pups compared to wild-type litter and age-matched controls. These results demonstrate the pathological potential of the mGluR5 knockout in rats and further support the presence of neuroinflammatory roots in ASDs.

Control of Fibrotic Gene Expression in Cardiac Myocytes and Fibroblasts via the Rap1 GTPase

Fibrosis describes the excessive deposition of extracellular matrix components by cardiac myofibroblasts (CFs), a process which frequently promotes the adverse effects of pathological conditions damaging cardiac muscle, such as myocardial infarction and arrhythmogenic cardiomyopathy. Roughly 1 million Americans suffer a myocardial infarction each year, after which necrotic cardiac myocytes (CMs) are replaced by CF‐derived extracellular matrix (ECM), in response to (a) biochemical signaling cues from necrotic CMs and (b) mechanical cues from the contracting myocardium and ventricle cavity pressure. Accumulation of ECM proteins (such as collagen) within the infarct scar is initially critical to prevent further tissue damage, but chronic and excessive collagen deposition is detrimental to regeneration of CMs and healing of the myocardium. While the physiological outcomes of cardiac fibrosis are well studied, the biochemical and mechano‐responsive signaling mechanisms which regulate fibrotic gene expression remain poorly investigated. The RhoA GTPase signaling pathway has been shown to play a central role in regulating fibrotic gene expression and is activated via many different pro‐fibrotic signaling factors and biomechanical stimuli (i.e., changes in ECM composition, organization and rigidity). The GTPase Rap1 is also very well‐known to mediate both inside‐out and outside‐in signaling via attachment of integrins to ECM proteins. In certain contexts, Rap1 has also been shown to antagonize RhoA signaling, but very little is known about its role in fibrotic gene expression. We therefore hypothesized that expression of Rap1 would inhibit fibrotic gene expression in cardiac myocytes and fibroblasts. Expression of either wildtype (WT) or constitutively active (63E) GFP‐tagged Rap1A constructs in cardiac fibroblasts resulted in a dramatic decrease in mRNA levels of ACTA2 (smooth muscle alpha‐actin), a commonly used marker of myofibroblast activation. Further, expression of WT or 63E Rap1A in either cardiac myocytes or fibroblasts resulted in a significant decrease in mRNA levels of ECM proteins (such as COL1A1, COL3A1, COL4A1 and FN1), as well as other common markers of fibrotic gene expression (CTGF and EGR1). Taken together, these data indicate that Rap1 signaling inhibits fibrotic gene expression and myofibroblast activation. Interestingly, expression of WT or 63E Rap1A in cardiac fibroblasts caused a dramatic increase in inflammatory markers (such as TNF, IL6, NFKB1A) and matrix remodeling enzymes (MMP3 and MMP9), genes which have been shown to play a role in increasing collagen turnover, reducing ECM rigidity/stiffness and repair of damaged myocardial tissue. Altogether, these data point to a significant role for Rap1 GTPase in reducing fibrotic gene expression and myofibroblast activation and suggest possible new therapeutic strategies to combat the damaging effects of cardiac fibrosis.

The potential inhibitory effect of ginsenoside Rh2 on mitophagy in UV-irradiated human dermal fibroblasts

BackgroundIn addition to its use as a health food, ginseng is used in cosmetics and shampoo because of its extensive health benefits. The ginsenoside, Rh2, is a component of ginseng that inhibits tumor cell proliferation and differentiation, promotes insulin secretion, improves insulin sensitivity, and shows antioxidant effects. MethodsThe effects of Rh2 on cell survival, extracellular matrix (ECM) protein expression, and cell differentiation were examined. The antioxidant effects of Rh2 in UV-irradiated normal human dermal fibroblast (NHDF) cells were also examined. The effects of Rh2 on mitochondrial function, morphology, and mitophagy were investigated in UV-irradiated NHDF cells. ResultsRh2 treatment promoted the proliferation of NHDF cells. Additionally, Rh2 increased the expression levels of ECM proteins and growth-associated immediate-early genes in ultraviolet (UV)-irradiated NHDF cells. Rh2 also affected antioxidant protein expression and increased total antioxidant capacity. Furthermore, treatment with Rh2 ameliorated the changes in mitochondrial morphology, induced the recovery of mitochondrial function, and inhibited the initiation of mitophagy in UV-irradiated NHDF cells. ConclusionRh2 inhibits mitophagy and reinstates mitochondrial ATP production and membrane potential in NHDF cells damaged by UV exposure, leading to the recovery of ECM, cell proliferation, and antioxidant capacity.

Potential role of noninvasive biomarkers during liver fibrosis.

Various types of liver disease exist, such as hepatitis and alcoholic liver disease. These liver diseases can result in scarring of liver tissue, cirrhosis, and finally liver failure. During liver fibrosis, there is an excess and disorganized accumulation of extracellular matrix (ECM) components which cause the loss of normal liver cell functions. For patients with chronic liver disease, fibrosis prediction is an essential part of the assessment and management. To diagnose liver fibrosis, several invasive and noninvasive markers have been proposed. However, the adoption of invasive markers remains limited due to their inherent characteristics and poor patient acceptance rate. In contrast, noninvasive markers can expedite the clinical decision through informed judgment about disease stage and prognosis. These noninvasive markers are classified into two types: Imaging techniques and serum biomarkers. However, the diagnostic values of biomarkers associated with liver fibrosis have also been analyzed. For example, the serum levels of ECM proteins can react to either matrix accumulation or degradation. During virus-host interactions, several regulatory steps take place to control gene expression, such as the change in cellular microRNA expression profiles. MicroRNAs are a class of non-coding RNAs (18-20 long nucleotides) that function by post-transcriptional regulation of gene expression. Although various noninvasive markers have been suggested in recent years, certain limitations have restricted their clinical applications. Understanding the potential of non-invasive biomarkers as a therapeutic option to treat liver fibrosis is still in progress.

Hopes and Hurdles of Employing Mesenchymal Stromal Cells in the Treatment of Cardiac Fibrosis.

Excessive cardiac fibrosis plays a crucial role in almost all types of heart disease. Generally, cardiac fibrosis is a scarring process triggered in response to stress, injury, or aging and is characterized by the accumulation of activated myofibroblasts that deposit high levels of extracellular matrix proteins in the myocardium. While it is beneficial for cardiac repair in the short term, it can also result in pathological remodeling, tissue stiffening, and cardiac dysfunction, contributing to the progression of heart failure, arrhythmia, and sudden cardiac death. Despite its high prevalence, there is a lack of effective and safe therapies that specifically target myofibroblasts to inhibit or even reverse pathological cardiac fibrosis. In the past few decades, cell therapy has been under continuous evaluation as a potential treatment strategy, and several studies have shown that transplantation of mesenchymal stromal cells (MSCs) can reduce cardiac fibrosis and improve heart function. Mechanistically, it is believed that the heart benefits from MSC therapy by stimulating innate anti-fibrotic and regenerative reactions. The mechanisms of action include paracrine signaling and cell-to-cell interactions. In this review, we provide an overview of the anti-fibrotic properties of MSCs and approaches to enhance them and discuss future directions of MSCs for the treatment of cardiac fibrosis.

The Role of Androgen Receptor in Cross Talk Between Stromal Cells and Prostate Cancer Epithelial Cells.

Prostate cancer (PCa) lists as the second most lethal cancer for men in western countries, and androgen receptor (AR) plays a central role in its initiation and progression, which prompts the development of androgen deprivation therapy (ADT) as the standard treatment. Prostate tumor microenvironment, consisting of stromal cells and extracellular matrix (ECM), has dynamic interactions with PCa epithelial cells and affects their growth and invasiveness. Studies have shown that both genomic and non-genomic AR signaling pathways are involved in the biological regulation of PCa epithelial cells. In addition, AR signaling in prostate stroma is also involved in PCa carcinogenesis and progression. Loss of AR in PCa stroma is clinically observed as PCa progresses to advanced stage. Especially, downregulation of AR in stromal fibroblasts dysregulates the expression levels of ECM proteins, thus creating a suitable environment for PCa cells to metastasize. Importantly, ADT treatment enhances this reciprocal interaction and predisposes stromal cells to promote cell invasion of PCa cells. During this process, AR in PCa epithelium actively responds to various stimuli derived from the surrounding stromal cells and undergoes enhanced degradation while elevating the expression of certain genes such as MMP9 responsible for cell invasion. AR reduction in epithelial cells also accelerates these cells to differentiate into cancer stem-like cells and neuroendocrine cells, which are AR-negative PCa cells and inherently resistant to ADT treatments. Overall, understanding of the cross talk between tumor microenvironment and PCa at the molecular level may assist the development of novel therapeutic strategies against this disease. This review will provide a snapshot of AR’s action when the interaction of stromal cells and PCa cells occurs.

INHIBITION OF BRD PROTEINS SUPPRESSES THE PHENOTYPE OF UTERINE FIBROIDS VIA REGULATION OF N6-METHYLADENOSINE REGULATORS

Bromodomain (BRD)-containing proteins are involved in many biological processes, most notably epigenetic regulation of transcription, and BRD protein dysfunction has been linked to many diseases including tumorigenesis. However, the role of BRD proteins in the pathogenesis of uterine fibroids (UFs), the most common pelvic tumors in women of reproductive age, is unknown. The present study aimed to determine the expression pattern of BRD proteins in UFs and matching myometrium and assess the effect of BRD inhibitors on UF phenotype and epitranscriptomic changes. Human UFs (n=22) and adjacent myometrium tissues (MyoF, n=7) were collected at the time of hysterectomy at the University of Chicago. Western blot (WB) was performed to determine the levels of BRD proteins in UFs and myometrium. Fourteen BRD inhibitors from the Epigenetic Chemical Probe Library provided by the University of Illinois at Chicago were used to determine the cell proliferation by MTT assay. Two BRD inhibitors (I-BRD9 and TP-472) were further used to explore their mechanistic actions. The student’s t-test was used to determine the significant difference. Our studies demonstrated that among 22 UFs analyzed, 96% (21/22, p<0.01), 55% (12/22), 82% (18/22, p<0.01) exhibited the upregulation of BRD2, BRD3, and BRD9 respectively, suggesting the aberrant BRD protein expression may contribute to the pathogenesis of UFs. To examine the biological impact of BRD inhibition on the UFs, 14 BRD inhibitors were screened to identify specific and potent BRD inhibitors in UF cells. 43% (6/14) of these BRD inhibitors selectively inhibited UF, but not myometrial cell proliferation. Furthermore, targeting BRD proteins with BRD inhibitors (I-BRD9 and TP-472) decreased the protein levels of extracellular matrix proteins (Type I Collagen, fibronectin), PCNA (cell proliferation marker), and Bcl-2 (anti-apoptotic marker) in a dose- and time-dependent manner in UF cells. Notably, inhibition of BRD proteins with I-BRD9 and TP-472 downregulated the expression of METTL3, YTHDC2, and YTHDF2, which are the key regulators for N6-methyladenosine (m6A) modification, indicating the tight link between BRD proteins and epitranscriptomic landscape. These results demonstrate for the first time that BRD proteins are aberrantly expressed in UFs. BRD inhibitors suppress the UF phenotype while concomitantly altering the expression of the central m6A regulators.