Fibroblast Biology Research Articles

Pathogenesis of fibrosis in interstitial lung disease

Pulmonary fibrosis is a chronic and progressive lung disease involving unclear pathological mechanisms. The present review presents and discusses the major and recent advances in our knowledge of the pathogenesis of lung fibrosis. The past months have deepened our understanding on the cellular actors of fibrosis with a better characterization of the abnormal lung epithelial cells observed during lung fibrosis. Better insight has been gained into fibroblast biology and the role of immune cells during fibrosis. Mechanistically, senescence appears as a key driver of the fibrotic process. Extracellular vesicles have been discovered as participating in the impaired cellular cross-talk during fibrosis and deeper understanding has been made on developmental signaling in lung fibrosis. This review emphasizes the contribution of different cell types and mechanisms during pulmonary fibrosis, highlights new insights for identification of potential therapeutic strategies, and underlines where future research is needed to answer remaining open questions.

Diversity and Biology of Cancer-Associated Fibroblasts.

Efforts to develop anti-cancer therapies have largely focused on targeting the epithelial compartment, despite the presence of non-neoplastic stromal components that substantially contribute to the progression of the tumor. Indeed, cancer cell survival, growth, migration, and even dormancy are influenced by the surrounding tumor microenvironment (TME). Within the TME, cancer-associated fibroblasts (CAFs) have been shown to play several roles in the development of a tumor. They secrete growth factors, inflammatory ligands, and extracellular matrix proteins that promote cancer cell proliferation, therapy resistance, and immune exclusion. However, recent work indicates that CAFs may also restrain tumor progression in some circumstances. In this review, we summarize the body of work on CAFs, with a particular focus on the most recent discoveries about fibroblast heterogeneity, plasticity, and functions. We also highlight the commonalities of fibroblasts present across different cancer types, and in normal and inflammatory states. Finally, we present the latest advances regarding therapeutic strategies targeting CAFs that are undergoing preclinical and clinical evaluation.

O30 Long non-coding RNA HOTAIR induces beta catenin expression in systemic sclerosis through EZH2 dependent repression of Axin-2

Abstract Background Fibroblasts explanted from affected tissues in systemic sclerosis (SSc) maintain their pro-fibrotic phenotype in vitro. This includes increased secretion of collagens and other extracellular matrix proteins and increased proportion of α-Smooth Muscle Actin (α-SMA) positive cells (myofibroblasts). It has been previously shown that among their profibrotic features, myofibroblasts display activation of WNT signalling, which is linked to a decreased basal expression of AXIN2. Here we aimed to determine whether specific long non-coding RNA (lncRNAs) expressed in myofibroblasts could drive the epigenetically stable decreased expression of Axin 2 in vitro. Methods Full thickness skin biopsies were surgically obtained from the forearms of twelve adult patients with SSc of recent onset. Fibroblasts were isolated and cultured in monolayers and protein and RNA extracted from the fibroblast cultures. Laser capture was performed to isolate cells expressing or not α-SMA as a marker of myofibroblast differentiation. LncRNA HOTAIR was overexpressed in healthy dermal fibroblasts by lentiviral induction. EZH2 was blocked in cultured fibroblasts with the specific inhibitor GSK126. Results HOTAIR expression was increased in SSc patients’ skin (100 fold) and in SSc explanted fibroblasts (5 fold; p < 0.001 for both). Further, laser captured α-SMA expressing fibroblasts expressed in average 2.5 fold higher HOTAIR RNA levels compared to α -SMA negative cells from the same donors (P < 0.05). In vitro, lentiviral induced stable overexpression of HOTAIR in healthy dermal fibroblasts led to their profibrotic activation, including significantly increased expression of Col1A1 and α-SMA both at mRNA and protein levels (2.8 and 1.8 fold respectively, p < 0.05). We further showed that HOTAIR-induced profibrotic activation was due to EZH2 dependent spread of H3k27me3 methylation marker, as demonstrated by complete inhibition by treatment with GSK126. HOTAIR driven EZH2 histone methylation suppressed the expression of Axin 2 in SSc fibroblasts. The reduced Axin 2 levels led to stabilisation of beta catenin and WNT signalling pathway activation. Conclusion Here we show that the epigenetically stable activation of SSc dermal fibroblasts is due to HOTAIR. We also identified a major downstream target of HOTAIR is Axin-2. The results of these studies identify a new venue to modulate fibroblasts biology which could inform clinical research to resolve chronic fibrosis and re-establish tissue homeostasis in SSc. Disclosures C.W. Wasson None. G. Abignano None. R. Ross None. F. Del Galdo Consultancies; AstraZeneca, GSK, Boehringer-Ingelheim, Actelion, Capella Biosciences, Chemomab.

Effect of endothelial dysfunction in hypertrophic scars on fibroblasts biology

Effect of endothelial dysfunction in hypertrophic scars on fibroblasts biology

Molecular biology of periodontal ligament fibroblasts and orthodontic tooth movement : Evidence and possible role of the circadian rhythm.

The circadian clock plays an important role in many physiological states and pathologies. The significance of its core genes in bone formation and tooth development has already been demonstrated. However, regulation of these genes and their influence on periodontal and bone remodeling in periodontal ligament (PDL) fibroblasts remains to be elucidated. Our hypothesis was that the circadian clock influences markers for periodontal and bone remodeling and therefore orthodontic tooth movement itself. Human PDL fibroblasts were cultured and synchronized in circadian rhythms with the help of adexamethasone shock. Cells were harvested at 4 h intervals. Reverse transcription and quantitative RT PCR (real time polymerase chain reaction) were performed to assess the mRNA levels of the clock genes ARNTL, CLOCK1, PER1, and PER2. Subsequently, mRNA expression of important marker genes for periodontal and bone remodeling, OPG, RANKL, OCN, OPN, RUNX2, COL1A1, IL1β, KI67, and POSTN, were examined at time points of ARNTL amplitude expression. Gene expression of core clock genes varied over 48 h in accordance with the circadian rhythm. Functional markers, except KI67, showed significant differences at time points of maximum fluctuation especially of ARNTL. PDL fibroblasts express circadian clock genes. Our results suggest that genes associated with bone and periodontal remodeling are influenced by the circadian rhythm. Further research will have to refine the understanding of this influence for orthodontic treatment.

Abstract 522: IL-10 Knockout Bone Marrow Fibroblast Progenitor Cells-derived Exosomes Activate Cardiac Fibroblast and Exaggerate Pressure Overload-induced Fibrosis in Mice Heart

Background: Inflammatory mediators play important role in cardiac remodeling. In heart, fibrosis is mainly mediated by activated fibroblasts (myofibroblasts), however, origin of myofibroblasts in diseased heart remains unclear. Recently, we have shown that bone marrow fibroblast progenitor cells (FPCs) significantly contribute in cardiac fibrosis in IL10 KO (KO) mice, but its molecular mechanism is not known. Here, we hypothesize that BM-FPCs activate resident cardiac fibroblast via their paracrine mediators (packaged in exosomes) to exaggerate the cardiac fibrosis in KO mice post- transverse aortic constriction (TAC). Method and Results: Cardiac hypertrophy and fibrosis was induced in Wild-type (WT) and IL10-knockout (IL10KO) mice by TAC surgery. TAC-induced left ventricular (LV) dysfunction and fibrosis were further exaggerated in KO mice. Recombinant IL10 administration markedly improved LV function and inhibited PO-induced cardiac fibrosis. PO enhanced FPC mobilization and homing in KO mice compared to WT mice. Furthermore, to identify the paracrine signaling, exosomes were isolated from WT and KO-FPCs culture media and characterized for fibrotic miRNA and proteins. Our data suggests that KO-FPC-exosomes are enriched with profibrotic miRs (miR21 and miR27) and proteins (RhoA). To explore whether KO-FPC-exosomes modulates resident fibroblasts biology and activation, adult cardiac fibroblasts (WT) were isolated and treated with WT and IL10KO-BMFPC-exosomes for 48 hrs. myoFB-associated gene (Collagen1α, Collagen3α and α- SMA) expression was notably increased in KO-FPC-exosome treated cells. Finally, immunostaining data further corroborated with our gene expression data and showed significantly enhanced α-SMA expression after KO-FPCs exosome treatments in cFBs. Conclusion: In conclusion, our data suggest that fibrotic remodeling factors (miRs and/or proteins) packaged in KO-FPC exosomes are sufficient to enhance the resident cardiac fibroblast activation and mediate cardiac fibrotic remodeling. Acknowledgement: NIH (RO1-135060) and AHA (14SDG20480104) SKV

Vitamin D and Wnt3A have additive and partially overlapping modulatory effects on gene expression and phenotype in human colon fibroblasts

The Wnt/β-catenin signalling pathway is essential for intestinal epithelium homeostasis, but its aberrant activation is a hallmark of colorectal cancer (CRC). Several studies indicate that the bioactive vitamin D metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) inhibits proliferation and promotes epithelial differentiation of colon carcinoma cells in part through antagonism of the Wnt/β-catenin pathway. It is now accepted that stromal fibroblasts are crucial in healthy and pathologic intestine: pericryptal myofibroblasts are constituents of the stem cell niche and cancer-associated fibroblasts (CAFs) contribute to CRC progression. However, studies on the combined action of 1,25(OH)2D3 and Wnt factors in colon fibroblasts are lacking. Here we show by global transcriptomic studies that 1,25(OH)2D3 and Wnt3A have profound, additive, partially overlapping effects on the gene expression profile of CCD-18Co human colon myofibroblasts. Moreover, 1,25(OH)2D3 and Wnt3A inhibit CCD-18Co cell proliferation and migration, while 1,25(OH)2D3 reduces, but Wnt3A increases, their capacity to contract collagen gels (a marker of fibroblast activation). These data were largely confirmed in patient-derived primary colon normal fibroblasts and CAFs, and in fibroblasts from other origins. Our results indicate that 1,25(OH)2D3 and Wnt3A are strong regulators of colon fibroblast biology and contribute to a better knowledge of intestinal homeostasis and stromal fibroblast action in CRC.

Inhibition of circHIPK3 prevents angiotensin II-induced cardiac fibrosis by sponging miR-29b-3p

BackgroundCircular RNAs (circRNAs) are emerging as powerful regulators of cardiac development and disease. Nevertheless, detailed studies describing circRNA-mediated regulation of cardiac fibroblasts (CFs) biology and their role in cardiac fibrosis remain limited. MethodsPCR and Sanger sequencing were performed to identify the expression of circHIPK3 in CFs. Edu corporation assays, Transwell migration assays, and immunofluorescence staining assays were conducted to detect the function of circHIPK3 in CFs in vitro. Bioinformatics analysis, dual luciferase activity assays, RNA immunoprecipitation, and fluorescent in situ hybridization experiments were conducted to investigate the mechanism of circHIPK3-mediated cardiac fibrosis. Echocardiographic analysis, Sirius Red staining and immunofluorescence staining were performed to investigate the function of circHIPK3 in angiotensin II (Ang II) induced cardiac fibrosis in vivo. ResultscircHIPK3 expression markedly increased in CFs and heart tissues after the treatment of Ang II. circHIPK3 silencing attenuates CFs proliferation, migration and the upregulation of a-SMA expression levels induced by Ang II in vitro. circHIPK3 acted as a miR-29b-3p sponge and overexpression of circHIPK3 effectively reverses miR-29b-3p-induced inhibition of CFs proliferation and migration and alters the expression levels of miR-29b-3p targeting genes (a-SMA, COL1A1, COL3A1) in vitro. Combination of circHIPK3 silencing and miR-29b-3p overexpression had a stronger effect on cardiac fibrosis suppression in vivo than did circHIPK3 silencing or miR-29b-3p overexpression alone. ConclusionsOur data suggest that circHIPK3 serves as a miR-29b-3p sponge to regulate CF proliferation, migration and development of cardiac fibrosis, revealing a potential new target for the prevention of Ang II-induced cardiac fibrosis.

Increased Baseline YAP/TAZ Nuclear Localization and Altered Transcriptional Activity in Uterine Fibroids

BackgroundUterine leiomyomas, also known as fibroids (UF), are benign neoplasms of the uterus. UF consist of smooth muscle cells and fibroblasts, and display enriched extracellular matrix (ECM). Reports estimate a cumulative incidence of 70% in premenopausal women, with annual total health care costs attributed to UF exceeding 34 billion dollars in the United States alone. In spite of their prevalence, the underlying processes that drive UF development and growth are still poorly understood. Accumulating evidence implicates the canonical Hippo signaling pathway effectors Yes‐associated protein (YAP)/transcriptional coactivator with PDZ‐binding domain (TAZ) as key drivers of fibroblast biology. Thus, we seek to assess the differences in YAP/TAZ localization, ECM deposition, and gene expression between UF and normal myometrium (Myo).MethodsMatched samples of UF and Myo were obtained from premenopausal women with IRB approval. Immunohistochemistry (N=5) was performed on OCT embedded tissue with YAP/TAZ antibody. For cell culture, UF and Myo were minced and digested with collagenase type 4. Cells (passages 1–4) were plated on plastic at confluence (N=4 for both). Immunostaining for YAP/TAZ, Fibronectin (FN), Collagen Type I (COL1), and Collagen Type III (COL3) were performed (N=4–5). Transcript levels were analyzed by treating cells with control or YAP/TAZ siRNA, and performing subsequent cDNA synthesis and qPCR reactions (N=4). Kruskall‐Wallis and Mann Whitney tests were used as appropriate.ResultsYAP/TAZ nuclear localization was upregulated in UF when compared to Myo tissue by immunofluorescence imaging. In freshly isolated cells cultured at confluence on tissue culture plastic, baseline YAP/TAZ nuclear localization was increased in UF when compared to Myo (p=0.03). UFs also exhibited a trend toward increased FN, COL1, and COL3 deposition when compared to Myo. UF displayed increased transcript levels for several matrix and pro‐fibrotic signaling genes (COL1A1, COL3A1, CTGF, EDN1, FN1) compared to the matched Myo. Following siRNA treatment for YAP/TAZ gene expression was reduced 40% and 50% for EDN1 (encoding endothelin‐1), as well as 70% and 65% for CTGF (encoding connective tissue growth factor), in UF and Myo respectively.ConclusionsUF nuclear YAP/TAZ is increased at baseline in vivo as well as in vitro. UF also display upregulated deposition of the ECM proteins. Additionally, there are increased transcript levels of profibrotic genes in UF compared to Myo. Moreover, the observed decreases in gene expression following siRNA knockdown of YAP/TAZ suggests a role for the Hippo signaling pathway in regulation of certain profibrotic genes in UF. Together these results suggest that enhanced baseline YAP/TAZ localization in UF may drive upregulated expression of pro‐fibrotic genes promoting UF progression.Support or Funding InformationCenter for Biomedical Discovery‐Department of Obstetrics and Gynecology Team Science Pilot Award ProgramThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of regulators of the myofibroblast phenotype of primary dermal fibroblasts from early diffuse systemic sclerosis patients

Systemic sclerosis (SSc or scleroderma) is an auto-immune disease characterized by skin fibrosis. While primary cells from patients are considered as a unique resource to better understand human disease biology, the effect of in vitro culture on these cells and their evaluation as a platform to identify disease regulators remain poorly characterized. The goal of our studies was to provide insights into the utility of SSc dermal fibroblast primary cells for therapeutic target discovery. The disease phenotypes of freshly isolated and in vitro cultured SSc dermal fibroblasts were characterized using whole transcriptome profiling, alpha smooth muscle actin (ASMA) expression and cell impedance. SSc dermal fibroblasts retained most of the molecular disease phenotype upon in vitro culture for at least four cell culture passages (approximatively 10 cell doublings). We validated an RNA interference high throughput assay that successfully identified genes affecting the myofibroblast phenotype of SSc skin fibroblasts. These genes included MKL1, RHOA and LOXL2 that were previously proposed as therapeutic anti-fibrotic target, and ITGA5, that has been less studied in fibrosis biology and may be a novel potential modifier of SSc fibroblast biology. Together our results demonstrated the value of carefully-phenotyped SSc dermal fibroblasts as a platform for SSc target and drug discovery.

Therapeutic interleukin-6 blockade reverses transforming growth factor-beta pathway activation in dermal fibroblasts: insights from the faSScinate clinical trial in systemic sclerosis

ObjectivesSkin fibrosis mediated by activated dermal fibroblasts is a hallmark of systemic sclerosis (SSc), especially in the subset of patients with diffuse disease. Transforming growth factor-beta (TGFβ) and interleukin-6 (IL-6)...

Lysophosphatidic Acid Regulates the Vitamin D Receptor and its Metabolism‐Related Genes in Human Gingival Fibroblasts

Our laboratory has established that lysophosphatidic acid (LPA) regulates the biology of human oral fibroblasts, and that gingival fibroblasts (GF) express the LPA1–5 receptor subtypes. We have also shown that LPA 18:1 controls immediate‐early GF gene transcription of inflammatory cytokines, and of their receptors and regulators. Vitamin D3 is a key hormone in decreasing inflammation, and has been shown by Nastri et al. (2018) to decrease pro‐inflammatory and increase anti‐inflammatory cytokine production by P. gingivalis‐ and S. pyogenes‐infected human GF. Therefore, we hypothesized that LPA would also modulate transcription of its receptor, VDR, since chronic periodontal disease (PD) is an inflammatory condition in which we have reported that LPA is elevated in human gingival crevicular fluid and saliva (Bathena et al., 2011). After GF treatment with 1 × 10−5 M LPA 18:1 for 2h or 8h, mRNA was extracted. Agilent Whole Human Genome Oligonucleotide Microarray analyses [n=3; each n= pooled mRNA derived from 3 healthy young donors; (9 total donors; the study conformed to the Declaration of Helsinki guidelines, was approved by the Creighton University IRB, and informed written consent was obtained from all donors)] revealed that LPA consistently and significantly down‐regulated the VDR vs. control (≤ −2.0 ± 0.5‐fold at 2h, and −3.1 ± 1.1 fold at 8h, respectively). Of great interest, the receptor cubilin (CUBN), which has been shown in other systems to bind vitamin D binding protein (DBP), was also down‐regulated in groups 1 (−2.0) and 2 (−4.1) at 8h; it may be involved in the secretion of vitamin D by GF, as postulated by Liu et al., (2012). LPA treatment also significantly affected transcription of the cytochromes (CYP) CYP27A1 [−3.0 ± 0.6 (2h); −3.2 ± 1.4 (8h)] and CYP27B1 (induction or repression varied by group) which is involved in the production of metabolically active metabolites and which catalyzes the hydroxylation at the C‐1α position of 25(OH)D3 and 24R, 25(OH)2D3, respectively. These results for the VDR (minimum level of significance, p < 1×10−7) provide preliminary evidence that LPA likely exerts complex actions during periodontal inflammation by regulating it, along with other key receptors and enzymes in the vitamin D pathway.Support or Funding InformationHealth Future Foundation (D.R.C).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolic and Phenotypic Characterization of Human Skin Fibroblasts After Forcing Oxidative Capacity.

Human skin fibroblasts present technical advantages for the study of mitochondrial-induced toxicity, because those cells can be isolated from patients by lowly invasive methods and present specific cumulative cellular damage and mutations of particular conditions. Several drugs lead to organ toxicity, with some of these drugs having been already withdrawn from the market. Frequently, drug-induced toxicity is attributed to mitochondrial liabilities. One of the approaches to identify drug-induced mitochondrial toxicity is using glucose-free/galactose/glutamine/pyruvate-containing cell culture media that force cells to be more dependent on oxidative phosphorylation for energy production. However, the effects of this modified culture medium itself on the mitochondrial phenotype of human skin fibroblasts have not been explored in detail. Our objective was to assess the mitochondrial biology of human skin fibroblasts under standard or modified culture conditions so that system can be validated and used in a more reliable way to disclose mitochondrial liabilities of drug candidates or intrinsic metabolic differences in fibroblasts. Our results showed that forcing mitochondrial remodeling in human skin fibroblasts increased oxygen consumption rate, ATP levels, and mitochondria-related transcripts and proteins. Moreover, the metabolic remodeling increased cytotoxicity of mitochondrial poisons. In general, no alterations in gene expression related with differentiation status were observed in human skin fibroblasts, with exception of increased paxilin gene expression. Not only the current work highlights the importance of using human skin primary cells to study drug-induced mitochondrial toxicity, it also reinforces the use of this tool to detect specific mitochondrial defects in skin fibroblasts from patients.

Advances in the Application and Impact of MicroRNAs as Therapies for Skin Disease.

The advent of RNA interference (RNAi) technology has profoundly impacted molecular biology research and medicine but has also advanced the field of skin care. Both effector molecules of RNAi, short-interfering RNA molecules and microRNAs (miRNAs), have been explored for their relative impact and utility for treating a variety of skin conditions. These post-transcriptional RNA regulatory molecules down-modulate protein expression through targeting of the 3′ untranslated regions of messenger RNAs, leading to their degradation or repression through sequestration. As researchers hunt for genetic linkages to skin diseases, miRNA regulators have emerged as key players in the biology of keratinocytes, fibroblasts, melanocytes, and other cells of the skin. Herein, we attempt to coalesce the current efforts to combat various skin disorders and diseases through the development of miRNA-based technologies.

Abstract 98: Canonical TGF-β1 Signaling in Cardiomyocytes is Essential to Maintain Basal Cardiac Function

The role of canonical transforming growth factor-β (TGF-β) pathway is well recognized in fibroblast biology and fibrosis in diseased hearts. However, its role in cardiomyocyte (CM) biology is not clear. SMAD4 is the central intracellular mediator of canonical TGF-β signaling. Herein, we investigate the role of SMAD4 in CM-biology and cardiac pathophysiology. SMAD4 homozygous floxed ( SMAD4 fl/fl ) and heterozygous floxed ( SMAD4 fl/- ) mice were crossed with α-myosin heavy chain (Mer-Cre-Mer) to create conditional CM-specific SMAD4-KO ( SMAD4 fl/fl Cre +/- ) and SMAD4 haploinsufficiency ( SMAD4 fl/- Cre +/- ), respectively. At 10 Wks of age, mice were subjected to well established tamoxifen diet protocol for 2 Wks. Echocardiographic analysis at 4 Wks post-tamoxifen treatment reveals that CM-specific loss of SMAD4 leads to dilatative ventricular remodeling as reflected by significantly increased LVIDs. This dilatative remodeling was associated with severe ventricular dysfunction as reflected by significantly reduced ejection fraction (EF) and fractional shortening (FS). Both HW/BW and LW/BW were significantly elevated in SMAD4 KO mice, suggesting pathological hypertrophy and heart failure in KOs. Analysis of Masson trichrome stained heart sections reveals a marked increase in fibrosis in the KO hearts. Q-PCR analysis showed the re-expression of fetal gene program (ANP, BNP), further confirming pathological remodeling in the KO hearts. As the heart functions of heterozygous mice and littermate controls were comparable at baseline, we stress them with TAC surgery. Consistent with KO findings, at 4 Wks post-TAC, heterozygous mice demonstrated characteristic heart failure phenotype. Taken together, these findings suggest that SMAD4 is required to maintain basal cardiac function and to prevent adverse ventricular remodeling in a pressure-overloaded heart.

Characterization and Standardization of Cultured Cardiac Fibroblasts for Ex Vivo Models of Heart Fibrosis and Heart Ischemia.

A full understanding of cardiac fibroblast (cFB) biology is essential to study the adverse cardiac remodeling and recovery of myocardium infarction. However, compared to cardiac myocytes, cFBs are less well characterized. Important questions, including the variability introduced by cell age (neonatal vs. adult), culture conditions (passage, plate coating, and culture medium), and responses to stimuli (e.g., hypoxia and drug treatments), have not been well addressed and standardization of techniques is lacking. This variability invites inconsistency and the confounding of study conclusions. Thus, we here focus on characterizing cell responses and standardizing procedures for cFB isolation and culture conditions to provide reliable platforms to address important questions about cFB proliferation, activation, collagen matrix formation, and responses to relevant stimuli. Thirty litters of 1-3-day pups and 30 female (240-330 g) Sprague-Dawley rats were used to isolate neonatal and adult cFBs. We detail and validate procedures to isolate cFBs for the use of culture or direct analysis. We characterize the differences between neonatal and adult cFBs, define the changes of cFBs during serial passage, and identify the response of cFBs to different culture conditions. We have also established models for the functional screening of profibrotic and antifibrotic drugs based on cFB proliferation, myofibroblast activation, and pericellular collagen matrix formation, and models of hypoxia/reoxygenation with appropriate time course and media conditions to achieve consistent cell injury. Our standardized procedures will ensure consistency in assessing cFB function. This original contribution provides a valid platform for the ex vivo investigation of the role of cFBs in cardiac ischemia and fibrosis.

Hypertrophic scar regression is linked to the occurrence of endothelial dysfunction.

Most microvessels have been shown to become stenosed or completely occluded during hypertrophic scar progression. Here, we examined the morphology of capillary endothelial cells (ECs) and fibroblasts using immunofluorescence staining for CD31 and alpha-smooth muscle actin (α-SMA) and electron microscopy. In addition, ECs and fibroblasts were isolated from scar tissues, and the levels of transforming growth factor beta 1 (TGF-β1), platelet-derived growth factor (PDGF), endothelin 1 (ET-1), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were assayed using ELISAs. Furthermore, we assessed cell viability, total collagen production, and cell apoptosis in hypertrophic scar-derived fibroblasts cultured with EC-conditioned medium. Then, anti-TGF-β1, anti-PDGF, anti-ET-1, anti-VEGF, and anti-bFGF neutralising antibodies were individually added to the EC medium to identify which growth factor plays a more important role in inhibiting fibroblasts biology. Our results showed microvessel lumen occlusion and EC atrophy during scar development, particularly in regressive scars (RSs). Additionally, EC growth factor secretion decreased and reached the lowest levels in RSs. Furthermore, based on the culture results, RS EC medium inhibited fibroblast viability and collagen production and induced apoptosis. Moreover, TGF-β1, PDGF, and bFGF played more important roles in these processes than VEGF and ET-1. The endothelial dysfunction occurring in hypertrophic scars contributes to fibroblast inhibition and scar regression, and reduced TGF-β1, PDGF, and bFGF levels play key roles during this process.

Mechanisms of Fibroblast Activation in the Remodeling Myocardium.

Activated fibroblasts are critically implicated in repair and remodeling of the injured heart. This manuscript discusses recent progress in the cell biology of fibroblasts in the infarcted and remodeling myocardium, highlighting advances in understanding the origin, function and mechanisms of activation of these cells. Following myocardial injury, fibroblasts undergo activation and myofibroblast transdifferentiation. Recently published studies have suggested that most activated myofibroblasts in the infarcted and pressure-overloaded hearts are derived from resident fibroblast populations. In the healing infarct, fibroblasts undergo dynamic phenotypic alterations in response to changes in the cytokine milieu and in the composition of the extracellular matrix. Fibroblasts do not simply serve as matrix-producing cells, but may also regulate inflammation, modulate cardiomyocyte survival and function, mediate angiogenesis, and contribute to phagocytosis of dead cells. In the injured myocardium, fibroblasts are derived predominantly from resident populations and serve a wide range of functions.

Properties and Immune Function of Cardiac Fibroblasts.

This chapter will discuss the role of cardiac fibroblasts as a target of various immunological inputs as well as an immunomodulatory hub of the heart through interaction with immune cell types and chemokine or cytokine signaling. While the purpose of this chapter is to explore the immunomodulatory properties of cardiac fibroblasts, it is important to note that cardiac fibroblasts are not a homogeneous cell type, but have a unique embryological origin and molecular identity. Specific properties of cardiac fibroblasts may influence the way they interact with the heart microenvironment to promote healthy homeostatic function or respond to pathological insults. Therefore, we will briefly discuss these aspects of cardiac fibroblast biology and then focus on their immunomodulatory role in the heart.

The biology and function of fibroblasts in cancer.

Among all cells, fibroblasts could be considered the cockroaches of the human body. They survive severe stress that is usually lethal to all other cells, and they are the only normal cell type that can be live-cultured from post-mortem and decaying tissue. Their resilient adaptation may reside in their intrinsic survival programmes and cellular plasticity. Cancer is associated with fibroblasts at all stages of disease progression, including metastasis, and they are a considerable component of the general host response to tissue damage caused by cancer cells. Cancer-associated fibroblasts (CAFs) become synthetic machines that produce many different tumour components. CAFs have a role in creating extracellular matrix (ECM) structure and metabolic and immune reprogramming of the tumour microenvironment with an impact on adaptive resistance to chemotherapy. The pleiotropic actions of CAFs on tumour cells are probably reflective of them being a heterogeneous and plastic population with context-dependent influence on cancer.