Increased Collagen Secretion Research Articles

Role of the mitochondrial protein cyclophilin D in skin wound healing and collagen secretion.

Central for wound healing is the formation of granulation tissue, which largely consists of collagen and whose importance stretches past wound healing, including being implicated in both fibrosis and skin aging. Cyclophilin D (CyD) is a mitochondrial protein that regulates the permeability transition pore, known for its role in apoptosis and ischemia-reperfusion. To date, the role of CyD in human wound healing and collagen generation has been largely unexplored. Here, we show that CyD was upregulated in normal wounds and venous ulcers, likely adaptive as CyD inhibition impaired reepithelialization, granulation tissue formation, and wound closure in both human and pig models. Overexpression of CyD increased keratinocyte migration and fibroblast proliferation, while its inhibition reduced migration. Independent of wound healing, CyD inhibition in fibroblasts reduced collagen secretion and caused endoplasmic reticulum collagen accumulation, while its overexpression increased collagen secretion. This was confirmed in a Ppif-KO mouse model, which showed a reduction in skin collagen. Overall, this study revealed previously unreported roles of CyD in skin, with implications for wound healing and beyond.

Single-cell Transcriptomic Analysis Reveals an Immunosuppressive Network Between POSTN CAFs and ACKR1 ECs in TKI-resistant Lung Cancer.

Tyrosine kinase inhibitor (TKI) therapy, a principal treatment for advanced non-small cell lung cancer (NSCLC), frequently encounters the development of drug resistance. The tumor microenvironment (TME) plays a critical role in the progression of NSCLC, yet the relationship between endothelial cells (ECs) and cancer-associated fibroblasts (CAFs) subpopulations in TKI treatment resistance remains largely unexplored. The BioProject database PRJNA591860 project was used to analyze scRNA-seq data including 49 advanced-stage NSCLC samples across three different time points: pre-targeted therapy (naïve), post-partial response (PR) to targeted therapy, and post-progressive disease (PD) stage. The data involved clustering stromal cells into multiple CAFs and ECs subpopulations. The abundance changes and functions of each cluster during TKI treatment were investigated by KEGG and GO analysis. Additionally, we identified specific transcription factors and metabolic pathways via DoRothEA and scMetabolism. Moreover, cell-cell communications between PD and PR stages were compared by CellChat. ECs and CAFs were clustered and annotated using 49 scRNA-seq samples. We identified seven ECs subpopulations, with OIT3 ECs showing enrichment in the PR phase with a drug-resistance phenotype, and ACKR1 ECs being prevalent in the PD phase with enhanced cell adhesion. Similarly, CAFs were clustered into 7 subpopulations. PLA2G2A CAFs were predominant in PR, whereas POSTN CAFs were prevalent in PD, characterized by an immunomodulatory phenotype and increased collagen secretion. CellChat analysis showed that ACKR1 ECs strongly interacted with macrophage through the CD39 pathway and POSTN CAFs secreted Tenascin-C (TNC) to promote the progression of epithelial cells, primarily malignant ones, in PD. This study reveals that POSTN CAFs and ACKR1 ECs are associated with resistance to TKI treatment, based on single-cell sequencing.

A new starch based wound dressing modified by graphene oxide/silver nanowire nanoparticles

Considering the special effect of wound treatment in the field of medicine, antibacterial wound dressings preparation is one of the main challenges in successful wound healing. In this research, a starch (St)/polyvinyl alcohol (PVA) nanofiberous mat modified with graphene oxide (GO)-silver nanowire (AgNW) hybrid was fabricated. Initially, silver nanowires were synthesized by the polyol method, then GO-AgNW nanoparticle was prepared as a biocompatible and antibacterial biofilm. Reducing the diameter of nanofibers due to the addition of starch and GO-AgNW nanoparticles leads to fibroblast proliferation, increased collagen secretion, reduced inflammation and wound closure. Nanofibrous mats showed significant antibacterial activity against Staphylococcus aureus and Escherichia coli microorganisms. The largest zone of bacterial inhibition was obtained for Starch/PVA nanofibers containing 8% GO-AgNWs hybrid (27.11 and 24.62 mm). The use of GO-AgNW nanoparticles without drugs is an important point due to the strong antibacterial property of silver nanowire. The measured contact angle of nanofibrous mats was in the range of 57.8° as a desirable level of hydrophilicity for wound dressings. The degree of swelling obtained about 491.12%, and the water vapor transmission rate of 2549 ± 36 (g/m2.day) as a proof for accelerating wound healing. Finally, the results indicated that St/PVA/GO-AgNWs composite electrospun fibers could be a suitable candidate for the treatment of infectious wounds, surgical wounds, minor burns, etc.

Cell differentiation and matrix organization are differentially affected during bone formation in osteogenesis imperfecta zebrafish models with different genetic defects impacting collagen type I structure

Osteogenesis imperfecta (OI) is a family of rare heritable skeletal disorders associated with dominant mutations in the collagen type I encoding genes and recessive defects in proteins involved in collagen type I synthesis and processing and in osteoblast differentiation and activity. Historically, it was believed that the OI bone phenotype was only caused by abnormal collagen type I fibrils in the extracellular matrix, but more recently it became clear that the altered bone cell homeostasis, due to mutant collagen retention, plays a relevant role in modulating disease severity in most of the OI forms and it is correlated to impaired bone cell differentiation. Despite in vitro evidence, in vivo data are missing. To better understand the physiopathology of OI, we used two zebrafish models: Chihuahua (Chi/+), carrying a dominant p.G736D substitution in the α1 chain of collagen type I, and the recessive p3h1−/−, lacking prolyl 3-hydroxylase (P3h1) enzyme. Both models share the delay of collagen type I folding, resulting in its overmodification and partial intracellular retention. The regeneration of the bony caudal fin of Chi/+ and p3h1−/− was employed to investigate the impact of abnormal collagen synthesis on bone cell differentiation. Reduced regenerative ability was evident in both models, but it was associated to impaired osteoblast differentiation and osteoblastogenesis/adipogenesis switch only in Chi/+. On the contrary, reduced osteoclast number and activity were found in both models during regeneration. The dominant OI model showed a more detrimental effect in the extracellular matrix organization. Interestingly, the chemical chaperone 4-phenylbutyrate (4-PBA), known to reduce cellular stress and increase collagen secretion, improved bone formation only in p3h1−/− by favoring caudal fin growth without affecting bone cell markers expression. Taken together, our in vivo data proved the negative impact of structurally abnormal collagen type I on bone formation but revealed a gene mutation-specific effect on bone cell differentiation and matrix organization in OI. These, together with the distinct ability to respond to the chaperone treatment, underline the need for precision medicine approaches to properly treat the disease.

Incorporation of strontium-containing bioactive particles into PEOT/PBT electrospun scaffolds for bone tissue regeneration

The combination of biomaterials and bioactive particles has shown to be a successful strategy to fabricate electrospun scaffolds for bone tissue engineering. Among the range of bioactive particles, hydroxyapatite and mesoporous bioactive glasses (MBGs) have been widely used for their osteoconductive and osteoinductive properties. Yet, the comparison between the chemical and mechanical characteristics as well as the biological performances of these particle-containing scaffolds have been characterized to a limited extent. In this work, we fabricated PEOT/PBT-based composite scaffolds incorporating either nanohydroxyapatite (nHA), strontium-containing nanohydroxyapatite (nHA_Sr) or MBGs doped with strontium ions up to 15 wt./vol% and 12,5 wt./vol% for nHA and MBG, respectively. The composite scaffolds presented a homogeneous particle distribution. Morphological, chemical and mechanical analysis revealed that the introduction of particles into the electrospun meshes caused a decrease in the fiber diameter and mechanical properties, yet maintaining the hydrophilic nature of the scaffolds. The Sr2+ release profile differed according to the considered system, observing a 35-day slowly decreasing release from strontium-containing nHA scaffolds, whereas MBG-based scaffolds showed a strong burst release in the first week. In vitro, culture of human bone marrow-derived mesenchymal stromal cells (hMSCs) on composite scaffolds demonstrated excellent cell adhesion and proliferation. In maintenance and osteogenic media, all composite scaffolds showed high mineralization as well as expression of Col I and OCN compared to PEOT/PBT scaffolds, suggesting their ability to boost bone formation even without osteogenic factors. The presence of strontium led to an increase in collagen secretion and matrix mineralization in osteogenic medium, while gene expression analysis showed that hMSCs cultured on nHA-based scaffolds had a higher expression of OCN, ALP and RUNX2 compared to cells cultured on nHA_Sr scaffolds in osteogenic medium. Yet, cells cultured on MBGs-based scaffolds showed a higher gene expression of COL1, ALP, RUNX2 and BMP2 in osteogenic medium compared to nHA-based scaffolds, which is hypothesized to lead to high osteoinductivity in long term cultures.

Collagen scaffold impregnated with borosilicate bioactive glass for endometrial healing

Artificial scaffolds that mimic the extracellular matrix (ECM) have promising applications in treating Asherman syndrome. In this study, we synthesized a bioactive glass (BG) by using the sol-gel method, and composite scaffolds with four different collagen concentrations (1, 2.5, 5 and 7.5 mg/mL) by freeze-drying approach. Next, we synthesized a novel collagen/BG composite by impregnating BG particles on the collagen scaffolds (CSBG1, CSBG2.5, CSBG5 and CSBG7.5 respectively). Mechanical analysis showed that the elastic modulus of CSBG5 reached 1 MPa which is comparable to soft tissues. Immunohistochemistry (IHC) results show that BG leads to a 1.93-fold increase in collagen secretion and a 3-fold increase in vascular endothelial growth factor (VEGF) secretion compared to the control groups. BG improves angiogenesis by a 4.9-fold increase in the number of branching points and a 6.5-fold increase in tube length. Immunofluorescence staining results revealed that CSBG5 has the greatest potential to be used as an endometrium scaffold since it has the highest cell viability and adhesion. Additional evidence includes increases in epithelial thickness, cytokeratin 7 (CK7) expressions, and the number of glands. Also, we observed that CSBG5 decreased calcification and inflammation. This study suggests that CSBG5 is a potential scaffold for uterine regeneration.

Decellularized Spinach Biomaterials Support Physiologically Relevant Mechanical Cyclic Strain and Prompt a Stretch-Induced Cellular Response.

Recently, decellularized plant biomaterials have been explored for their use as tissue engineered substitutes. Herein, we expanded upon the investigation of the mechanical properties of these materials to explore their elasticity as many anatomical areas of the body require biomechanical dynamism. We first constructed a device to secure the scaffold and induce a strain within the physiological range of the normal human adult lung during breathing (12-20 movements/min; 10-20% elongation). Results showed that decellularized spinach leaves can support cyclic strain for 24 h and displayed heterogeneous local strain values (7.76-15.88%) as well as a Poisson's ratio (0.12) similar to that of mammalian lungs (10.67-19.67%; 0.01), as opposed to an incompressible homogeneous standard polymer (such as PDMS (10.85-12.71%; 0.4)). Imaging and mechanical testing showed that the vegetal scaffold exhibited strain hardening but maintained its structural architecture and water retention capacity, suggesting an unaltered porosity. Interestingly, we also showed that cells seeded on the scaffold can also sense the mechanical strain as demonstrated by a nuclear reorientation perpendicular to strain direction (63.3° compared to 41.2° for nonstretched cells), a nuclear location of YAP and increased expression of YAP target genes, a high cytoplasmic calcium level, and an elevated expression level of collagen genes (COL1A1, COL3A1, COL4A1, and COL6A) with an increased collagen secretion at the protein level. Taken together, these data demonstrated that decellularized plant leaf tissues have an inherent elastic property similar to that found in the mammalian system to which cells can sense and respond.

Volatile Constituents and Anti-Osteoporotic Activity of the n-Hexane Extract From Homalomena gigantea Rhizome

This study analyzed the chemical composition and anti-osteoporosis activity of the n-hexane extract of Homalomena gigantea rhizome. Sixty compounds, representing 92.0% of the extract, were identified by gas chromatography-mass spectrometry. Linalool (15.3%), oplopanone (9.8%), ( Ε)-α-atlantone (5.6%), khusinol acetate (5.4%), bullatantriol (4.3%), and β-sitosterol (3.8%) were the main constituents. The anti-osteoporotic activity of the n-hexane extract was determined by measuring alkaline phosphatase (ALP) activity, collagen content, and the mineralization of MC3T3-E1 cells. At concentrations of 4.0 and 20.0 µg/mL, the n-hexane extract increased ALP activity by 8.2% and 23.7%, and increased collagen secretion by MC3T3-E1 cells by 114.9% and 112.4%, respectively. At 4 µg/mL, the extract significantly promoted the mineralization of MC3T3-E1 cells by as much as 133.2% compared to the negative control. These results suggested that H. gigantea rhizome contains a natural anti-osteoporotic compound.

Tricomposite gelatin-carboxymethylcellulose-alginate bioink for direct and indirect 3D printing of human knee meniscal scaffold

The development of technologies that could ease the production of customizable patient-specific tissue engineering constructs having required biomechanical properties and restoring function in damaged tissue is the need of the hour. In this study, we report the optimization of composite, bioactive and biocompatible tripolymeric hydrogel bioink, suitable for both direct and indirect printing of customizable scaffolds for cartilage tissue engineering applications. A customized hierarchical meniscal scaffold was designed using solid works software and developed using a negative mould made of polylactic acid (PLA) filament and by a direct 3D printing process. A composite tripolymeric bioink made of gelatin, carboxymethyl cellulose (CMC) and alginate was optimized and characterized for its printability, structural, bio-mechanical and bio-functional properties. The optimized composite hydrogel bioink was extruded into the negative mould with and without live cells, cross-linked and the replica of meniscus structure was retrieved aseptically. The cellular proliferation, apatite formation, and extracellular matrix secretion from negative printed meniscal scaffold were determined using MTT, live/dead and collagen estimation assays. A significant increase in collagen secretion, cellular proliferation and changes in biomechanical properties was observed in the 3D scaffolds with MG63-osteosarcoma cells indicating its suitability for cartilage tissue engineering.

Viability and collagen secretion by fibroblasts on titanium surfaces with different acid-etching protocols

BackgroundFrom the consolidation of surface treatments of dental implants and knowledge on the cellular mechanisms of osseointegration, studies have highlighted the importance of a connective tissue seal against the implant to prevent contamination from the oral environment and consequent biofilm formation.ObjectiveThis in vitro study aimed to evaluate whether different titanium surface treatments using acid solutions promoted an increase in collagen secretion, proliferation, and viability of fibroblasts.Material and methodsCommercially pure grade-4 titanium disks (6 × 2 mm) were treated with different acid solutions (hydrochloric, nitric, and sulfuric) for 20 and 60 min, respectively, obtaining mean surface roughness of 0.1 to 0.15 μm and 0.5 to 0.7 μm. Human fibroblasts were seeded onto different surfaces and assessed after 24 h, 48 h, and 72 h for cell proliferation and viability using Trypan blue staining and MTT, respectively, as well as the secretion of type I collagen on to such surfaces using ELISA. Machined titanium surfaces were used as controls. Data were statistically analyzed using one-way ANOVA and Fisher's LSD test for multiple comparisons, adopting a significance level of 5%.ResultsNo significant difference was observed in cell proliferation for the different surfaces analyzed. Cell viability was significantly lower on the machined surface, after 48 h, when compared to the groups treated with acid for 20 or 60 min, which did not differ from each other. The expression of type I collagen was lowest on the acid-treated surfaces.ConclusionThe results showed that the acid treatment proposed did not promote fibroblast proliferation and viability nor favor type I collagen synthesis.

Cadherin-11 Is a Regulator of Intestinal Fibrosis

Although the mechanisms underlying the formation of intestinal fibrostrictures in Crohn's disease [CD] are not fully understood, activation of fibroblasts and excessive collagen deposition are supposed to contribute to the development of such complications. Here, we investigated the role of cadherin-11 [CDH-11], a fibroblast-derived protein that induces collagen production in various organs, in intestinal fibrosis. CDH-11 expression was evaluated in inflammatory [I] and fibrostricturing [FS] CD mucosal samples, ulcerative colitis [UC] mucosal samples, and ileal and colonic control samples, by real-time polymerase chain reaction, western blotting, and immunohistochemistry. CDH-11 expression was evaluated in normal and in CD intestinal fibroblasts stimulated with inflammatory/fibrogenic cytokines. FS CD fibroblasts were cultured either with a specific CDH-11 antisense oligonucleotide [AS], or activating CDH-11 fusion protein and activation of RhoA/ROCK, and TGF-β pathways and collagen production were evaluated by western blotting. Finally, we assessed the susceptibility of CDH-11-knockout [KO] mice to colitis-induced intestinal fibrosis. CDH-11 RNA and protein expression were increased in both CD and UC as compared with controls. In CD, the greater expression of CDH-11 was seen in FS samples. Stimulation of fibroblasts with TNF-α, interleukin [IL]-6, IFN-γ, IL-13, and IL-1β enhanced CDH-11 expression. Knockdown of CDH-11 in FS CD fibroblasts impaired RhoA/ROCK/TGF-β signalling and reduced collagen synthesis, whereas activation of CDH-11 increased collagen secretion. CDH-11 KO mice were largely protected from intestinal fibrosis. Data show that CDH-11 expression is up-regulated in inflammatory bowel disease [IBD] and suggest a role for this protein in the control of intestinal fibrosis.

Deletion of Calcineurin Promotes a Protumorigenic Fibroblast Phenotype.

Fibroblast activation is a crucial step in tumor growth and metastatic progression. Activated fibroblasts remodel the extracellular matrix (ECM) in primary tumor and metastatic microenvironments, exerting both pro- and antitumorigenic effects. However, the intrinsic mechanisms that regulate the activation of fibroblasts are not well-defined. The signaling axis comprising the calcium-activated Ser/Thr phosphatase calcineurin (CN), and its downstream target nuclear factor of activated T cells, has been implicated in endothelial (EC) and immune cell activation, but its role in fibroblasts is not known. Here, we demonstrate that deletion of CN in fibroblasts in vitro altered fibroblast morphology and function consistent with an activated phenotype relative to wild-type fibroblasts. CN-null fibroblasts had a greater migratory capacity, increased collagen secretion and remodeling, and promoted more robust EC activation in vitro. ECM generated by CN-null fibroblasts contained more collagen with greater alignment of fibrillar collagen compared with wild-type fibroblast-derived matrix. These differences in matrix composition and organization imposed distinct changes in morphology and cytoskeletal architecture of both fibroblasts and tumor cells. Consistent with this in vitro phenotype, mice with stromal CN deletion had a greater incidence and larger lung metastases. Our data suggest that CN signaling contributes to the maintenance of fibroblast homeostasis and that loss of CN is sufficient to promote fibroblast activation. SIGNIFICANCE: Calcineurin signaling is a key pathway underlying fibroblast homeostasis that could be targeted to potentially prevent fibroblast activation in distant metastatic sites.

Macrophage-secreted TGF-β1 contributes to fibroblast activation and ureteral stricture after ablation injury.

Iatrogenic injury to the healthy ureter during ureteroscope-guided ablation of malignant or nonmalignant disease can result in ureteral stricture. Transforming growth factor (TGF)-β1-mediated scar formation is considered to underlie ureteral stricture, but the cellular sources of this cytokine and the sequelae preceding iatrogenic stricture formation are unknown. Using a swine model of ureteral injury with irreversible electroporation (IRE), we evaluated the cellular sources of TGF-β1 and scar formation at the site of injury and examined in vitro whether the effects of TGF-β1 could be attenuated by pirfenidone. We observed that proliferation and α-smooth muscle actin expression by fibroblasts were restricted to injured tissue and coincided with proliferation of macrophages. Collagen deposition and scarring of the ureter were associated with increased TGF-β1 expression in both fibroblasts and macrophages. Using in vitro experiments, we demonstrated that macrophages stimulated by cells that were killed with IRE, but not LPS, secreted TGF-β1, consistent with a wound healing phenotype. Furthermore, using 3T3 fibroblasts, we demonstrated that stimulation with paracrine TGF-β1 is necessary and sufficient to promote differentiation of fibroblasts and increase collagen secretion. In vitro, we also showed that treatment with pirfenidone, which modulates TGF-β1 activity, limits proliferation and TGF-β1 secretion in macrophages and scar formation-related activity by fibroblasts. In conclusion, we identified wound healing-related macrophages to be an important source of TGF-β1 in the injured ureter, which may be a paracrine source of TGF-β1 driving scar formation by fibroblasts, resulting in stricture formation.

36 - Biogenic aldehydes influence extracellular matrix via fibroblast-mediated and collagen cross-linking mechanisms: Potential therapeutic role for RAGE antagonists and L-carnosine

Biogenic aldehydes are toxic byproducts of normal cellular metabolism and are known to contribute to chronic diseases. Oxidative deamination of norepinephrine and dopamine by monoamine oxidase (MAO) generates the ‘catecholaldehydes’ DOPEGAL and DOPAL, respectively, and H2O2. 4-Hydroxynonenal (HNE) is formed by oxidation of polyunsaturated fatty acids. These highly reactive electrophiles have been implicated as causal factors in the etiology of neurodegenerative diseases and cardiac injury from ischemia and diabetes. In diabetics, we and others have documented increased aldehyde accumulation and carbonyl stress in the heart. A pathogenic role for these aldehydes in cardiac fibrosis has been proposed, but mechanisms by which they act in this manner remain unclear. We hypothesize that aldehydes act via two pathways- 1) by activating fibroblasts via receptor for advanced glycation endproducts (RAGE)-dependent mechanisms; and 2) by cross-linking extracellular matrix proteins. Treatment of cardiac fibroblasts with DOPAL-BSA conjugate resulted in a >2-fold increase in collagen secretion and a robust increase in reactive oxygen species (ROS), and these effects were abolished by FPS-ZM1, a RAGE antagonist. To explore non-cellular mechanisms of aldehyde’s role in fibrosis, collagen was incubated with DOPEGAL, DOPAL and 4HNE alone or in presence of a concentrated glucose solution (25 mM) to simulate hyperglycemia. Yet another set of these incubations were repeated in presence of 5 mM solution of carnosine, a dipeptide that detoxifies aldehydes via formation of benign adducts . As determined by the shear modulus within the visco-elastic range of the polymer, we found that when combined together, aldehydes catalyze collagen cross-linking, and specifically catecholaldehydes enhanced the collagen crosslinking effects of glucose (P

Abstract 111: Reduced MK5 Expression Alters Migration, Proliferation, and Collagen Biosynthesis in Murine Ventricular Fibroblasts

MAP kinase-activated protein kinase-5 (MK5), a protein serine/threonine kinase expressed in the heart, has been identified as a substrate for p38α/β and ERK3/4 MAPKs. However, the interacting partners and physiological function of MK5 are just beginning to be understood. This study examined the role of MK5 in murine cardiac ventricular fibroblasts. Confocal immunocytofluorescence microscopy (CIFM) revealed that MK5 immunoreactivity localized primarily to the nucleus whereas ERK3 immunoreactivity was in the cytoplasm. Following serum stimulation, phospho-MK5 immunoreactivity was observed in the cytoplasm and appeared to be associated with the cytoskeleton and pseudopodia. ERK3 immunoreactivity redistributed to membrane ruffles and/or lamellipodia. ERK3 has been reported to be unstable in the absence of MK5: in cardiac fibroblasts, ERK3 immunoreactivity was unaffected by acute knockdown of MK5 with siRNA (MK5-kd), suggesting an as-yet unidentified binding partner may stabilize ERK3 in these cells. MK5 immunoprecipitates from fibroblast lysates contained ERK3 immunoreactivity and proximity ligation assays indicated the presence of ERK3-MK5 complexes in the cytoplasm: these complexes were less abundant in MK5-kd fibroblasts. Cell migration, in response to serum and/or Ang-II was reduced significantly in MK5-kd fibroblasts. In addition, cell proliferation was decreased in fibroblasts isolated from MK5 -/- mice compared to fibroblasts from wild-type litter mate mice (MK5 +/+ ). Surprisingly, both the abundance of type 1 collagen (COL1A1) mRNA and the secretion of soluble COL1A1 were increased in MK5-kd fibroblasts whereas the ability of Ang-II to increase collagen secretion was unaffected. CIFM revealed staining for COL1A1 was diffuse in MK5 +/+ fibroblasts but condensed in the perinuclear region of MK5 -/- cells. A similar pattern of subcellular distribution of COL1A1 immunoreactivity was observed in MK5-kd fibroblasts and was observed in passages 0 through 3. Taken together, these data suggest 1) one or more proteins in addition to MK5 serve to stabilize ERK3 in fibroblasts and 2) MK5 may be involved in cardiac fibroblast proliferation, migration, collagen biosynthesis and, possibly, myocardial remodelling.

The reactive dopamine metabolite 3,4‐Dihydroxyphenylacetaldehyde (DOPAL) is a potent inducer of collagen secretion in human cardiac fibroblasts

Spontaneous generation of reactive sugar‐ and lipid‐derived aldehydes occurs in vivo as a byproduct of oxidative stress, and these aldehydes form protein adducts via electrophilic attack on cysteine and lysine residues. Catecholaldehydes are biogenic aldehydes formed as products of catecholamine metabolism by monoamine oxidase (MAO). MAO catalyzes the oxidative deamination of dopamine to generate 3,4‐dihydroxyphenylacetaldehyde (DOPAL), a reactive molecule that has been shown to be neurotoxic stemming from the reactivity of both catechol and aldehyde groups. Our lab has recently reported that diabetic patients have higher content and activity of MAO in atrial myocardium as compared with age‐matched nondiabetic patients, and that MAO activity in this tissue is associated with postoperative atrial fibrillation. Increased collagen deposition in the heart (i.e., fibrosis) is a well‐known risk factor for arrhythmias, and is also associated with diabetes. Here, we tested the hypothesis that DOPAL‐adducts induce production and secretion of collagen in human cardiac fibroblasts. Fibroblasts were isolated and cultured from right atrial appendage samples obtained from patients during during cardiac surgery. Cells were then treated with bovine serum albumin (BSA) conjugated with DOPAL, N(6)‐Carboxymethyllysine (CML, an advanced glycation end product), and 4‐hydroxynonenal (HNE, a n6 polyunsaturated fatty acid‐derived aldehyde). Collagen type I and III were measured in the media via immunoblot. Collagen content in the media increased with CML‐, HNE‐ and DOPAL‐BSA (P<0.05), as compared with BSA alone (control). Surprisingly, treatment with DOPAL increased type I and type III collagen secretion by 11‐fold and 32‐fold, respectively, compared to vehicle‐treated cells (P<0.05), an effect that was >5‐fold greater than with either CML or HNE. These findings suggest that reactive aldehydes, particularly catecholaldehydes, may significantly contribute to fibrosis in the heart via increased collagen secretion. Further, they provide evidence that compounds with aldehyde‐scavenging capacity may have therapeutic value in mitigating cardiac fibrosis.Support or Funding InformationR01HL122863‐01

MP17-09 CXCL12-CXCR4 AXIS ACTIVATION PROMOTES COP II VESICLE-MEDIATED SECRETION OF COLLAGEN BY PROSTATE MYOFIBROBLASTS

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology1 Apr 2017MP17-09 CXCL12-CXCR4 AXIS ACTIVATION PROMOTES COP II VESICLE-MEDIATED SECRETION OF COLLAGEN BY PROSTATE MYOFIBROBLASTS Susan Patalano-Salsman, Jose A. Rodriguez-Nieves, Diego Almanza, Amy Avery, Andrew Judell-Halfpenney, Todd Riley, and Jill Macoska Susan Patalano-SalsmanSusan Patalano-Salsman More articles by this author , Jose A. Rodriguez-NievesJose A. Rodriguez-Nieves More articles by this author , Diego AlmanzaDiego Almanza More articles by this author , Amy AveryAmy Avery More articles by this author , Andrew Judell-HalfpenneyAndrew Judell-Halfpenney More articles by this author , Todd RileyTodd Riley More articles by this author , and Jill MacoskaJill Macoska More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.598AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Factors that promote lower urinary tract voiding dysfunction (LUTD) in aging men include excessive prostatic proliferation and muscle contraction. Recent studies suggest that tissue fibrosis, e.g., peri-urethral collagen accumulation, may also contribute to LUTD. Our group recently reported that activation of the CXCL12/CXCR4 axis and downstream MEK/ERK activation, as well as canonical TGFβ/TGFβR axis activation and downstream Smad3 activation, promote prostate fibroblast to myofibroblast phenoconversion and collagen production. Based on this finding, we hypothesized that downstream MEK/ERK and Smad signaling would converge at the transcriptional level to promote the activation of genes encoding pro-fibrotic proteins. To test this, RNA sequencing analysis was performed on prostate fibroblasts treated with CXCL12 or TGFβ. METHODS Prostate fibroblasts were treated with 4ng/ml TGFβ or 100pM CXCL12 for 12 hrs (RNA) or 24, 48 and 72 hrs (protein). Total RNA was purified and subjected to qRT-PCR or RNASeq. Sequence data was pipelined through the Bowtie/TopHat/Cufflinks/CummeRbund (Tuxedo) packages. Pathview v1.10.1 and Cytoscape were used to perform Kegg pathway and network analysis, respectively. Immunoblot analysis was performed using whole protein lysates or concentrated conditioned media. RESULTS RNASeq analysis showed that prostate fibroblasts treated with CXCL12 up-regulated the transcript levels of genes that encoded members of the Cullin-RING 3 (CRL3) ubiquitin ligase family of proteins. These proteins form COPII vesicles that transport procollagen from the endoplasmic reticulum to the cell membrane and extracellular space. Western blot analysis showed that CXCL12-treated cells up-regulated CRL3 proteins and secreted higher levels of procollagen compared to vehicle and TGFβ-treated cells. Procollagen secretion was ablated upon treatment with AMD3100, a CXCR4 antagonist, demonstrating that the observed increased collagen secretion was specifically coupled to CXCL12/CXCR4 axis activation. CONCLUSIONS The results of these studies are consistent with our hypothesis and present a major new discovery: Activation of the CXCL12/CXCR4 axis promotes fibrosis by increasing both the expression and secretion of collagen. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e214-e215 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Susan Patalano-Salsman More articles by this author Jose A. Rodriguez-Nieves More articles by this author Diego Almanza More articles by this author Amy Avery More articles by this author Andrew Judell-Halfpenney More articles by this author Todd Riley More articles by this author Jill Macoska More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Enhanced store-operated Ca2+ influx and ORAI1 expression in ventricular fibroblasts from human failing heart.

ABSTRACTExcessive cardiac fibrosis, characterized by increased collagen-rich extracellular matrix (ECM) deposition, is a major predisposing factor for mechanical and electrical dysfunction in heart failure (HF). The human ventricular fibroblast (hVF) remodeling mechanisms that cause excessive collagen deposition in HF are unclear, although reports suggest a role for intracellular free Ca2+ in fibrosis. Therefore, we determined the association of differences in cellular Ca2+ dynamics and collagen secretion/deposition between hVFs from failing and normal (control) hearts. Histology of left ventricle sections (Masson trichrome) confirmed excessive fibrosis in HF versus normal. In vitro, hVFs from HF showed increased secretion/deposition of soluble collagen in 48 h of culture compared with control [85.9±7.4 µg/106 cells vs 58.5±8.8 µg/106 cells, P<0.05; (Sircol™ assay)]. However, collagen gene expressions (COL1A1 and COL1A2; RT-PCR) were not different. Ca2+ imaging (fluo-3) of isolated hVFs showed no difference in the thapsigargin-induced intracellular Ca2+ release capacity (control 16±1.4% vs HF 17±1.1%); however, Ca2+ influx via store-operated Ca2+ entry/Ca2+ release-activated channels (SOCE/CRAC) was significantly (P≤0.05) greater in HF-hVFs (47±3%) compared with non-failing (35±5%). Immunoblotting for ICRAC channel components showed increased ORAI1 expression in HF-hVFs compared with normal without any difference in STIM1 expression. The Pearson's correlation coefficient for co-localization of STIM1/ORAI1 was significantly (P<0.01) greater in HF (0.5±0.01) than control (0.4±0.01) hVFs. The increase in collagen secretion of HF versus control hVFs was eliminated by incubation of hVFs with YM58483 (10 µM), a selective ICRAC inhibitor, for 48 h (66.78±5.87 µg/106 cells vs 55.81±7.09 µg/106 cells, P=0.27). In conclusion, hVFs from HF have increased collagen secretion capacity versus non-failing hearts and this is related to increase in Ca2+ entry via SOCE and enhanced expression of ORAI, the pore-forming subunit. Therapeutic inhibition of SOCE may reduce the progression of cardiac fibrosis/HF.

Abstract 97: Ca 2+ Influx Via I CRAC Channels is Involved in Increased Secretion/Deposition of Collagen by Human Failing Heart Ventricular Fibroblasts

Introduction: Excessive cardiac fibrosis (CF) underlies mechanical and electrical dysfunction in heart failure (HF). However, the underlying mechanisms of increased CF are unclear. Recently, we reported increased Orai1 expression and I CRAC (calcium release-activated channels) -Ca 2+ influx in failing heart ventricular fibroblasts (hVF). Therefore, we studied the magnitude of collagen secretion/deposition by hVF from failing hearts and the involvement of Ca 2+ influx through I CRAC channels in this process. Hypothesis: We tested the hypothesis that collagen secretion/deposition by hVFs from failing hearts will be high and sensitive to inhibition of I CRAC channels, compared to normal hearts. Methods: Primary cultures of hVF obtained from HF patients undergoing LV assist device implantation (n=9) were compared to non-diseased hVF from trauma victims (n=4). The degree of fibrosis was analyzed by histology of LV tissue sections (5μm) with Masson’s trichrome staining. Collagen secretion/deposition after 48 h culture of hVFs from failing or normal hearts in the presence or absence of I CRAC inhibitor, YM-58483(10 μM), was assayed calorimetrically using Sircol™ kit. Data were analyzed by unpaired Student’s t-test. Results: Histology of the failing heart LV sections with Masson’s trichome staining showed severe fibrosis compared to control sections. The percentage of positive staining area to total area, measured using custom-developed ImageJ macro, was significantly (p&lt;0.05) higher in the failing heart (4.5±0.2%) compared to that of normal (0.1± 0.01%). The secretion/deposition of soluble collagen was significantly (p&lt;0.05) higher in failing heart hVF (86± 7.6 μg/10 6 cells) than normal heart hVF (58.4±8.8 μg/10 6 cells). Incubation of fibroblasts with the I CRAC inhibitor YM58486 (10 μM) for 48 h eliminated the increase in collagen secretion of HF fibroblasts (55.8± 7 μg/10 6 cells vs 66.8± 6 μg/10 6 cells, p=0.27). Conclusion: LV fibrosis is severe in HF patients. The magnitude of pro-fibrotic collagen secretion/deposition by hVF from HF patients is significantly high but sensitive to I CRAC inhibition. Selective targeting of fibroblast I CRAC may be a potential therapeutic tool to mitigate progression of CF and HF.

Substrate Stiffness and Microtopography in PIP2 Regulation of the Actin Cytoskeleton in Primary Cardiac Fibroblasts

Following tissue injury, fibroblasts are one of the primary responding cell types in the wound healing process. Upon myocardial infarction, local fibroblasts divide and increase collagen secretion, leading to cardiac fibrosis. In this study, the effect of the mechanical environment, through substrate stiffness and three‐dimensional topography, is examined in relation to the distribution and levels of phosphatidylinositol 4,5‐bisphosphate (PIP2) in primary neonatal rat ventricular fibroblasts. PIP2 is a membrane component and known regulator of the actin cytoskeleton. Substrates possessing 10 µm high microprojections are fabricated from polydimethylsiloxane to a stiffness of either 100 kPa or 400 kPa (Young's Modulus). A 3‐fold cellular decrease (P&lt;.001) in PIP2 is found between flat‐soft substrates and flat‐polystyrene culture dishes. Additionally, microprojections are shown to blunt PIP2 by a similar amount within each stiffness condition. Fluorescence recovery after photobleaching, yielding the rate constant Kfrap, allows study of actin dynamics in the various micromechanical conditions. A 4‐fold decrease (P&lt;.0001) is found between actin dynamics within lamellipodia to those within stress cables, correlating to a migrating or an anchored cell. This difference in dynamics coincides with an increased redistribution of PIP2 to the lamella in migrating cells. An improved understanding of the intracellular lipid mechanisms involved in wound healing could aid in prevention of maladaptive scarring following major injury. Funded by NIH HL62426, T32HL07692