3-D culture of human lung fibroblasts decreases proliferative and increases extracellular matrix remodeling genes.

Fibroblasts are the main producers of extracellular matrix (ECM) responsible for ECM maintenance and repair, a process often disrupted in chronic lung diseases. The accompanying mechanical changes adversely affect resident cells and overall lung function. Numerous models have been used to elucidate fibroblast behavior that are now evolving toward complex three-dimensional (3-D) models incorporating ECM, aiming to replicate the cells' native environment. Little is known about the cellular changes that occur when moving from two-dimensional (2-D) to 3-D cell culture. This study compared the gene expression profiles of primary human lung fibroblasts from seven subjects with normal lung function, that were cultured for 24 h on 2-D collagen I-coated tissue culture plastic and in 3-D collagen I hydrogels, which are commonly used to mimic ECM in various models, from contraction assays to intricate organ-on-a-chip models. Comparing 3-D with 2-D cell culture, 6,771 differentially expressed genes (2,896 up, 3,875 down) were found; enriched gene sets within the downregulated genes, identified through Gene Set Enrichment Analysis and Ingenuity Pathway Analysis, were involved in the initiation of DNA replication which implied downregulation of fibroblast proliferation in 3-D. Observation of cells for 72 h in 2-D and 3-D environments confirmed the reduced progression through the cell cycle in 3-D. A focused analysis, examining the Hippo pathway and ECM-associated genes, showed differential patterns of gene expression in the 3-D versus 2-D culture. Altogether, the transcriptional response of fibroblasts cultured in 3-D indicated inhibition of proliferation, and alterations in Hippo and ECM pathways indicating a complete switch from proliferation to ECM remodeling.NEW & NOTEWORTHY With the introduction of complex three-dimensional (3-D) lung models, comes a need for understanding cellular behavior in these models. We compared gene expression profiles of human lung fibroblasts grown on two-dimensional (2-D) collagen I-coated surfaces with those in 3-D collagen I hydrogels. RNA sequencing and subsequent pathway analyses showed decreased proliferation, increased extracellular matrix (ECM) remodeling, and altered Hippo signaling and ECM deposition-related gene signatures. These findings highlight unique responses of fibroblasts in 3-D models.

Regenerative treatments for scleroderma in cutaneous manifestations of the face: a systematic review.

OBJECTIVES: Scleroderma is a heterogeneous chronic autoimmune disease affecting connective tissue, characterised by chronic inflammation and fibrosis, particularly affecting internal organs and skin. Orofacial involvement is common, leading to facial atrophy, mask-like appearance and difficulties in function that significantly impact patients' quality of life. This systematic review evaluates different autologous regenerative treatments of facial manifestations of scleroderma, aiming to provide comprehensive understanding of their effectiveness in reducing fibrosis, and thereby improving function and skin quality. METHODS: A search in PubMed, Embase, Web of Science Core Collection, Cochrane CENTRAL, and CINAHL was conducted. Studies assessing autologous regenerative treatments in cutaneous manifestations of the face in scleroderma patients were included. Outcomes of interest were treatment characteristics, characterisation of biomaterials, outcome measurements and patient satisfaction. Methodological quality was assessed with the Effective Public Health Practice Project tool. RESULTS: In total 18 studies were included. Methodological quality of studies was weak (n=15) and moderate (n=3). Treatments consisted of autologous fat grafting, platelet-rich plasma, stromal vascular fraction, and adipose-derived stem cells. In general, most studies showed improvements of symptoms, but no treatment was considered superior. CONCLUSIONS: Autologous regenerative treatments hold potential for alleviating cutaneous manifestations of the face in scleroderma. Further clinical trials should be well-designed to improve the quality of clinical evidence.

Gene expression profiles in mesenchymal stromal cells from bone marrow, adipose tissue and lung tissue of COPD patients and controls.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by irreversible lung tissue damage. Novel regenerative strategies are urgently awaited. Cultured mesenchymal stem/stromal cells (MSCs) have shown promising results in experimental models of COPD, but differences between sources may impact on their potential use in therapeutic strategies in patients. AIM: To assess the transcriptome of lung-derived MSCs (LMSCs), bone marrow-derived MSCs (BM-MSC) and adipose-derived MSCs (AD-MSCs) from COPD patients and non-COPD controls. METHODS: We studied differences in gene expression profiles between the MSC-subtypes, as well as between COPD and control using RNA sequencing (RNA-seq). RESULTS: We show that besides heterogeneity between donors, MSCs from different sources have strongly divergent gene signatures. The growth factors FGF10 and HGF were predominantly expressed in LMSCs. MSCs from all sources displayed altered expression profiles in COPD, with most pronounced significantly up- and downregulated genes in MSCs from adipose tissue. Pathway analysis revealed that the most differentially expressed genes in COPD-derived AD-MSCs are involved in extracellular matrix (ECM) binding and expression. In LMSCs, the gene that differed most strongly between COPD and control was CSGALNACT1, an ECM modulating gene. CONCLUSION: Autologous MSCs from COPD patients display abnormalities with respect to their transcriptome, which were surprisingly most profound in MSCs from extrapulmonary sources. LMSCs may be optimally equipped for lung tissue repair because of the expression of specific growth factor genes.

Tissue Stromal Vascular Fraction Improves Early Scar Healing: A Prospective Randomized Multicenter Clinical Trial.

BACKGROUND: Wound healing and scar formation depends on a plethora of factors. Given the impact of abnormal scar formation, interventions aimed to improve scar formation would be most advantageous. The tissue stromal vascular fraction (tSVF) of adipose tissue is composed of a heterogenous mixture of cells embedded in extracellular matrix. It contains growth factors and cytokines involved in wound-healing processes, eg, parenchymal proliferation, inflammation, angiogenesis, and matrix remodeling. OBJECTIVES: The aim of this study was to investigate the hypothesis that tSVF reduces postsurgical scar formation. METHODS: This prospective, double-blind, placebo-controlled, randomized trial was conducted between 2016 and 2020. Forty mammoplasty patients were enrolled and followed for 1 year. At the end of the mammoplasty procedure, all patients received tSVF in the lateral 5 cm of the horizontal scar of 1 breast and a placebo injection in the contralateral breast to serve as an intrapatient control. Primary outcome was scar quality measure by the Patient and Observer Scar Assessment Scale (POSAS). Secondary outcomes were obtained from photographic evaluation and histologic analysis of scar tissue samples. RESULTS: Thirty-four of 40 patients completed follow-up. At 6 months postoperation, injection of tSVF had significantly improved postoperative scar appearance as assessed by the POSAS questionnaire. No difference was observed at 12 months postoperation. No improvement was seen based on the evaluation of photographs and histologic analysis of postoperative scars between both groups. CONCLUSIONS: Injection of tSVF resulted in improved wound healing and reduced scar formation at 6 months postoperation, without any noticeable advantageous effects seen at 12 months.

Supplementation of Facial Fat Grafting to Increase Volume Retention: A Systematic Review.

BACKGROUND: For decades, facial fat grafting has been used in clinical practice for volume restoration. The main challenge of this technique is variable volume retention. The addition of supplements to augment fat grafts and increase volume retention has been reported in recent years. OBJECTIVES: The aim of this systematic review was to investigate which supplements increase volume retention in facial fat grafting as assessed by volumetric outcomes and patient satisfaction. METHODS: Embase, Medline, Ovid, Web of Science Core Collection, Cochrane Central Register of Controlled Trials, and Google Scholar were searched up to November 30, 2020. Only studies assessing volume after facial fat grafting with supplementation in human subjects were included. Outcomes of interest were volume or patient satisfaction. The quality of the studies was assessed with the Effective Public Health Practice Project tool. RESULTS: After duplicates were removed 3724 studies were screened by title and abstract. After reading 95 full-text articles, 27 studies were eligible and included for comparison. Supplementation comprised of platelet-rich plasma, platelet-rich fibrin, adipose tissue-derived stromal cells or bone marrow-derived stromal cells, cellular or tissue stromal vascular fraction, or nanofat. In 13 out of 22 studies the supplemented group showed improved volumetric retention and 5 out of 16 studies showed greater satisfaction. The scientific quality of the studies was rated as weak for 20 of 27 studies, moderate for 6 of 27 studies, and strong for 1 study. CONCLUSIONS: It remains unclear if additives contribute to facial fat graft retention and there is a need to standardize methodology.

Autologous Lipofilling Improves Clinical Outcome in Patients With Symptomatic Dermal Scars Through Induction of a Pro-Regenerative Immune Response.

BACKGROUND: Autologous lipofilling is an emerging procedure to treat and possibly reverse dermal scars and to reduce scar-related pain, but its efficacy and mechanisms are poorly understood. OBJECTIVES: The aim of this study was to test the hypothesis that repeated lipografts reverse dermal scars by reinitiation of wound healing. METHODS: In a prospective, non-placebo-controlled clinical study, 27 adult patients with symptomatic scars were given 2 lipofilling treatments at 3-month intervals. As primary outcome, clinical effects were measured with the Patient and Observer Scar Assessment Scale (POSAS). Scar biopsies were taken before and after treatments to assess scar remodeling at a cellular level. RESULTS: Twenty patients completed the study. Patients' scars improved after lipofilling. The total POSAS scores (combined patient and observer scores) decreased from 73.2  [14.7] points (mean [standard deviation]) pretreatment to 46.1 [14.0] and 32.3 [13.2] points after the first and second lipofilling treatment, respectively. Patient POSAS scores decreased from 37.3 [8.8] points to 27.2 [11.3] and 21.1 [11.4] points, whereas observer POSAS scores decreased from 35.9 [9.5] points to 18.9 [6.0] and 11.3 [4.5] points after the first and second treatment, respectively. After each lipofilling treatment, T lymphocytes, mast cells, and M2 macrophages had invaded scar tissue and were associated with increased vascularization. In addition, the scar-associated epidermis showed an increase in epidermal cell proliferation to levels similar to that normal in skin. Moreover, lipofilling treatment caused normalization of the extracellular matrix organization towards that of normal skin. CONCLUSIONS: Autologous lipofilling improves the clinical outcome of dermal scars through the induction of a pro-regenerative immune response, increased vascularization, and epidermal proliferation and remodeling of scar tissue extracellular matrix.

The Addition of Tissue Stromal Vascular Fraction to Platelet-Rich Plasma Supplemented Lipofilling Does Not Improve Facial Skin Quality: A Prospective Randomized Clinical Trial.

BACKGROUND: Lipofilling has become popular as a treatment to improve aging-related skin characteristics (eg, wrinkles, pigmentation spots, pores, or rosacea). Different additives such as platelet-rich plasma (PRP) or stromal vascular fraction (SVF) have been combined with lipofilling to increase the therapeutic effect of adipose-derived stromal cells (ASCs). OBJECTIVES: The aim of this study was to examine the hypothesis that mechanically isolated SVF augments the therapeutic effect of PRP-supplemented lipofilling to improve facial skin quality. METHODS: This prospective, double-blind, placebo-controlled, randomized trial was conducted between 2016 and 2019. In total, 28 female subjects were enrolled; 25 completed the follow-up. All patients received PRP-supplemented lipofilling with either mechanically isolated SVF or saline. SVF was isolated by fractionation of adipose tissue (tSVF). Results were evaluated by changes in skin elasticity and transepidermal water loss, changes in skin-aging-related features, ie, superficial spots, wrinkles, skin texture, pores, vascularity, and pigmentation, as well as patient satisfaction (FACE-Q), recovery, and number of complications up to 1 year postoperative. RESULTS: The addition of tSVF to PRP-supplemented lipofilling did not improve skin elasticity, transepidermal water loss, or skin-aging-related features. No improvement in patient satisfaction with overall facial appearance or facial skin quality was seen when tSVF was added to PRP-supplemented lipofilling. CONCLUSIONS: In comparison to PRP-supplemented lipofilling, PRP-supplemented lipofilling combined with tSVF does not improve facial skin quality or patient satisfaction in a healthy population. PRP-supplemented lipofilling combined with tSVF can be considered a safe procedure.

MicroRNA-374b induces endothelial-to-mesenchymal transition and early lesion formation through the inhibition of MAPK7 signaling.

Endothelial-mesenchymal transition occurs during intimal hyperplasia and neointima formation via mechanisms that are incompletely understood. Endothelial MAPK7 signaling is a key mechanosensitive factor that protects against endothelial-mesenchymal transition, but its signaling activity is lost in vessel areas that are undergoing pathological remodeling. At sites of vascular remodeling in mice and pigs, endothelial MAPK7 signaling was lost. The TGFß-induced microRNA-374b targets MAPK7 and its downstream effectors in endothelial cells, and its expression induces endothelial-mesenchymal transition. Gain-of-function experiments, where endothelial MAPK7 signaling was restored, precluded endothelial-mesenchymal transition. In human coronary artery disease, disease severity is associated with decreased MAPK7 expression levels and increased miR-374b expression levels. Endothelial-mesenchymal transition occurs in intimal hyperplasia and early lesion formation and is governed in part by microRNA-374b-induced silencing of MAPK7 signaling. Restoration of MAPK7 signaling abrogated these pathological effects in endothelial cells expressing miR-374b. Thus, our data suggest that the TGFß-miR-374b-MAPK7 axis plays a key role in the induction of endothelial-mesenchymal transition during intimal hyperplasia and early lesion formation and might pose an interesting target for antiatherosclerosis therapy. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Fractionation of Adipose Tissue Procedure With a Disposable One-Hole Fractionator.

BACKGROUND: Adipose tissue has been widely used in regenerative surgery for its therapeutic potential. This potential is often ascribed to the stromal vascular fraction (SVF), which can be mechanically isolated. Mechanical isolation results in an SVF that retains intact cell-cell communication including extracellular matrix and is therefore named tissue-SVF (tSVF). OBJECTIVES: The aim of this study was to evaluate a new disposable 1-hole fractionator for fractionation of adipose tissue (FAT), and compare this new device with the existing reusable 3-hole fractionator. METHODS: The composition of tSVF obtained via the 1-hole fractionator was histologically and histochemically compared to unprocessed adipose tissue. The number of viable nuclear cells in tSVF obtained by the 1-hole and 3-hole fractionators as well as unprocessed adipose tissue were compared after enzymatic isolation and tested for colony-forming capacity. Flow cytometry was used to compare different cell compositions based on surface marker expression between tSVF isolated by the two types of fractionators. RESULTS: Fractionation of adipose tissue with the 1-hole fractionator condenses vasculature and extracellular matrix by disrupting adipocytes. The number of viable nuclear cells in tSVF obtained with the two fractionators was comparable and significantly higher than unprocessed lipoaspirate. Furthermore, tSVF isolated by both fractionators showed similar cell compositions and comparable colony-forming capacities. CONCLUSIONS: FAT with a disposable 1-hole fractionator effectively isolates tSVF with a cell count and cell composition comparable to the fraction obtained with the 3-hole reusable fractionator. The disposable 1-hole fractionator, however, is safer and more user friendly.

The Development of Facial Lipofilling from a Historical Point of View.

Lipofilling, the transplantation of adipose tissue, has already been used since the end of the 19th century. For decades, lipofilling was used to restore loss of volume due to aging, trauma, or congenital defects. Later on, the indications for the use of lipofilling expanded by treating aged skin, scars, and improving wound healing. The expansion was caused by the discovery of adipose derived stromal cells (ASCs) in adipose tissue and the development of very fine harvesting and injection cannulas which made it possible to inject small adipose tissue particles in small volume areas, such as the face. ASCs are multipotent stromal cells which reside in the stromal vascular fraction (SVF) of adipose tissue and are able to differentiate in multiple cell lineages and secrete a plurality of growth factors with regenerative potentials. The discovery of ASCs led toward more experimental cell-based therapies, that is, ASCs or SVF isolated by means of enzymatic isolation procedures. Later on, enzymatic isolation procedures were forbidden in many countries by legislation and were replaced by mechanical isolation procedures, such as the Nanofat and Fractionation of Adipose Tissue (FAT) procedures. The Nanofat procedure has been extensively investigated, especially as treatment for skin rejuvenation in the face. Though, substantial evidence is lacking for using facial lipofilling or any therapeutic component, that is, ASCs or SVF for skin rejuvenation to date. In contrast, facial lipofilling to restore loss of volume seems to be promising.

Human adipose tissue-derived stromal cells act as functional pericytes in mice and suppress high-glucose-induced proinflammatory activation of bovine retinal endothelial cells.

AIMS/HYPOTHESIS: The immunomodulatory capacity of adipose tissue-derived stromal cells (ASCs) is relevant for next-generation cell therapies that aim to reverse tissue dysfunction such as that caused by diabetes. Pericyte dropout from retinal capillaries underlies diabetic retinopathy and the subsequent aberrant angiogenesis. METHODS: We investigated the pericytic function of ASCs after intravitreal injection of ASCs in mice with retinopathy of prematurity as a model for clinical diabetic retinopathy. In addition, ASCs influence their environment by paracrine signalling. For this, we assessed the immunomodulatory capacity of conditioned medium from cultured ASCs (ASC-Cme) on high glucose (HG)-stimulated bovine retinal endothelial cells (BRECs). RESULTS: ASCs augmented and stabilised retinal angiogenesis and co-localised with capillaries at a pericyte-specific position. This indicates that cultured ASCs exert juxtacrine signalling in retinal microvessels. ASC-Cme alleviated HG-induced oxidative stress and its subsequent upregulation of downstream targets in an NF-κB dependent fashion in cultured BRECs. Functionally, monocyte adhesion to the monolayers of activated BRECs was also decreased by treatment with ASC-Cme and correlated with a decline in expression of adhesion-related genes such as SELE, ICAM1 and VCAM1. CONCLUSIONS/INTERPRETATION: The ability of ASC-Cme to immunomodulate HG-challenged BRECs is related to the length of time for which ASCs were preconditioned in HG medium. Conditioned medium from ASCs that had been chronically exposed to HG medium was able to normalise the HG-challenged BRECs to normal glucose levels. In contrast, conditioned medium from ASCs that had been exposed to HG medium for a shorter time did not have this effect. Our results show that the manner of HG preconditioning of ASCs dictates their immunoregulatory properties and thus the potential outcome of treatment of diabetic retinopathy.

The decrease in histone methyltransferase EZH2 in response to fluid shear stress alters endothelial gene expression and promotes quiescence.

High uniform fluid shear stress (FSS) is atheroprotective and preserves the endothelial phenotype and function through activation of downstream mediators such as MAPK7 (Erk5). Endothelial cells respond to FSS thanks to mechanotransduction. However, how the resulting signaling is integrated and resolved at the epigenetic level remains elusive. We hypothesized that Polycomb methyltransferase EZH2 is involved in the effects of FSS in human endothelial cells. We showed that FSS decreases the expression of the Polycomb methyltransferase EZH2. Despite simultaneous activation of MAPK7, MAPK7 pathway does not directly influence the transcription of EZH2. Interestingly though, the knockdown of EZH2 activates the protective MAPK7 signaling in endothelial cells, even in the absence of FSS. To understand the influence of the FSS-decreased expression of EZH2 on endothelial transcriptome, we performed RNA-seq and differential gene expression analysis. We identified candidate groups of genes dependent on both EZH2 and FSS. Among those, Gene Ontology overrepresentation analysis revealed highly significant enrichment of the cell cycle-related genes, suggesting changes in proliferation. Indeed, the depletion of EZH2 strongly inhibited endothelial proliferation, indicating cell cycle arrest. The concomitant decrease in CCNA expression suggests the transition of endothelial cells into a quiescent phenotype. Further bioinformatical analysis suggested TXNIP as a possible mediator between EZH2 and cell cycle-related gene network. Our data show that EZH2 is a FSS-responsive gene. Decreased EZH2 levels enhance the activation of the atheroprotective MAPK7 signaling. Decrease in EZH2 under FSS mediates the decrease in the expression of the network of cell cycle-related genes, which allows the cells to enter quiescence. EZH2 is therefore important for the protective effects of FSS in endothelium.

N-octanoyl dopamine treatment exerts renoprotective properties in acute kidney injury but not in renal allograft recipients.

BACKGROUND: N-octanoyl dopamine (NOD) treatment improves renal function when applied to brain dead donors and in the setting of warm ischaemia-induced acute kidney injury (AKI). Because it also activates transient receptor potential vanilloid type 1 (TRPV1) channels, we first assessed if NOD conveys its renoprotective properties in warm ischaemia-induced AKI via TRPV1 and secondly, if renal transplant recipients also benefit from NOD treatment. METHODS: We induced warm renal ischaemia in Lewis, wild-type (WT) and TRPV1(-/-) Sprague-Dawley (sd) rats by clamping the left renal artery for 45 min. Transplantations were performed in allogeneic and syngeneic donor-recipient combinations (Fisher to Lewis and Lewis to Lewis) with a cold ischaemia time of 20 h. Treatment was instituted directly after restoration of organ perfusion. Renal function, histology and perfusion were assessed by serum creatinine, microscopy and magnetic resonance imaging (MRI) using arterial spin labelling (ASL). RESULTS: NOD treatment significantly improved renal function in Lewis rats after warm ischaemia-induced AKI. It was, however, not effective after prolonged cold ischaemia. The renoprotective properties of NOD were only observed in Lewis or WT, but not in TRPV1(-/-) sd rats. Renal inflammation was significantly abrogated by NOD. MRI-ASL showed a significantly lower cortical perfusion in ischaemic when compared with non-ischaemic kidneys. No overall differences were observed in renal perfusion between NOD- and NaCl-treated rats. CONCLUSIONS: NOD treatment reduces renal injury in warm ischaemia, but is not effective in renal transplant in our experimental animal models. The salutary effect of NOD appears to be TPRV1-dependent, not involving large changes in renal perfusion.

The fractionation of adipose tissue procedure to obtain stromal vascular fractions for regenerative purposes.

Autologous adipose tissue transplantation is clinically used to reduce dermal scarring and to restore volume loss. The therapeutic benefit on tissue damage more likely depends on the stromal vascular fraction of adipose tissue than on the adipocyte fraction. This stromal vascular fraction can be obtained by dissociation of adipose tissue, either enzymatically or mechanical. Enzymatic dissociation procedures are time-consuming and expensive. Therefore, we developed a new inexpensive mechanical dissociation procedure to obtain the stromal vascular fraction from adipose tissue in a time sparing way, which is directly available for therapeutic injection. This mechanical dissociation procedure is denoted as the fractionation of adipose tissue (FAT) procedure. The FAT procedure was performed in eleven patients. The composition of the FAT-stromal vascular fraction was characterized by immunohistochemistry. Adipose derived stromal cells isolated from the FAT-stromal vascular fraction were compared with adipose derived stromal cells isolated from nondissociated adipose tissue (control) for their CD-surface marker expression, differentiation and colony forming unit capacity. Case reports demonstrated the therapeutic effect of the FAT-stromal vascular fraction. The FAT-stromal vascular fraction is an enrichment of extracellular matrix containing a microvasculature and culturable adipose derived stromal cells. Adipose derived stromal cells isolated from FAT-stromal vascular fraction did not differ from adipose derived stromal cells isolated from the control group in CD-surface marker expression, differentiation and colony forming unit capacity. The FAT procedure is a rapid effective mechanical dissociation procedure to generate FAT-stromal vascular fraction ready for injection with all its therapeutic components of adipose tissue: it contains culturable adipose derived stromal cells embedded in their natural supportive extracellular matrix together with the microvasculature.

H3K27Me3 abundance increases fibrogenesis during endothelial-to-mesenchymal transition via the silencing of microRNA-29c.

Objective: Endothelial-to-mesenchymal transition (EndMT) is a transdifferentiation process in which endothelial cells (ECs) adopt a mesenchymal-like phenotype. Over the past few years, it became clear that EndMT can contribute to several cardiovascular pathologies. However, the molecular pathways underlying the development of EndMT remain incompletely understood. Since the epigenetic enzyme Enhancer of Zeste Homolog 2 (EZH2) and its concomitant mark H3K27Me3 have been shown to be elevated in many cardiovascular diseases that associate with EndMT, we hypothesized that H3K27Me3 is a determinant for the susceptibility of EndMT. Methods: To study the association between H3K27Me3 and EndMT, a knockdown model of EZH2 in human endothelial cells (HUVEC) was utilized to reduce H3K27Me3 abundance, followed by induction of EndMT using TGFß1. The expression of molecular markers of EndMT and fibrogenesis were analysed. Results: In cultured HUVECs, a reduction of H3K27Me3 abundance facilitates EndMT but mitigates fibrogenesis as shown by a decreased expression of collagen I and III. In HUVEC, H3K27Me3 abundance directly affects the expression of miR29c, a collagen-targeting miRNA. Additionally, knockdown of miR-29c in HUVEC with low H3K27Me3 abundance partly restored the expression of collagen I and III. Expectedly, in rats with perivascular fibrosis an increased abundance of H3K27Me3 associated with a decreased expression of miR-29c. Conclusion: our data shows that endothelial fibrogenesis underlies an epigenetic regulatory pathway and we demonstrate that a decreased abundance of H3K27Me3 in ECs blunts fibrogenesis in part in a miR-29c dependent manner. Therefore, a reduction of H3K27Me3 could serve as a novel therapeutical strategy to mitigate fibrogenesis and may prove to be beneficial in fibrogenic diseases including atherosclerosis, cardiac fibrosis, and PAH.

The role of extracellular matrix hydrogels and adipose-derived stromal cells in soft tissue vascularization - A systematic review.

Decellularized extracellular matrix (dECM) hydrogels loaded with adipose-derived stromal cells (ASC) or their conditioned medium (ASC CM) present a promising and versatile treatment approach for tissue vascularization and regeneration. These hydrogels are easy to produce, store, personalize, manipulate, and deliver to the target tissue. This literature review aimed to investigate the applications of dECM hydrogels with ASC or ASC CM for in vivo tissue vascularization. Fourteen experimental studies have been reviewed using vessel density as the primary outcome parameter for in vivo vascularization. The studies consistently reported an increased efficacy in augmenting angiogenesis by the ASC or ASC CM-loaded hydrogels compared to untreated controls. However, this systematic review shows the need to standardize procedures and characterization, particularly of the final administered product(s). The findings from these experimental studies highlight the potential of dECM hydrogel with ASC or ASC CM in novel tissue regeneration and regenerative medicine applications.

Physical Properties and Biochemical Composition of Extracellular Matrix-Derived Hydrogels Dictate Vascularization Potential in an Organ-Dependent Fashion.

The inherent extracellular matrix (ECM) originating from a specific tissue impacts the process of vascularization, specifically vascular network formation (VNF) orchestrated by endothelial cells (ECs). The specific contribution toward these processes of ECM from highly disparate organs such as the skin and lungs remains a relatively unexplored area. In this study, we compared VNF and ECM remodeling mediated by microvascular ECs within gel, lung, and combinations thereof (hybrid) ECM hydrogels. Irrespective of the EC source, the skin-derived ECM hydrogel exhibited a higher propensity to drive and support VNF compared to both lung and hybrid ECM hydrogels. There were distinct disparities in the physical properties of the three types of hydrogels, including viscoelastic properties and complex architectural configurations, including fiber diameter, pore area, and numbers among the fibers. The hybrid ECM hydrogel properties were unique and not the sum of the component ECM parts. Furthermore, cellular ECM remodeling responses varied with skin ECM hydrogels promoting matrix metalloproteinase 1 (MMP1) secretion, while hybrid ECM hydrogels exhibited increased MMP9, fibronectin, and collagen IV deposition. Principal component analysis (PCA) indicated that the influence of a gel's mechanical properties on VNF was stronger than the biochemical composition. These data indicate that the organ-specific properties of an ECM dictate its capacity to support VNF, while intriguingly showing that ECs respond to more than just the biochemical constituents of an ECM. The study suggests potential applications in regenerative medicine by strategically selecting ECM origin or combinations to manipulate vascularization, offering promising prospects for enhancing wound healing through pro-regenerative interventions.

Fibroblast alignment and matrix remodeling induced by a stiffness gradient in a skin-derived extracellular matrix hydrogel.

Large skin injuries heal as scars. Stiffness gradually increases from normal skin to scar tissue (20x higher), due to excessive deposition and crosslinking of extracellular matrix (ECM) mostly produced by (myo)fibroblasts. Using a custom mold, skin-derived ECM hydrogels (dECM) were UV crosslinked after diffusion of ruthenium (Ru) to produce a Ru-dECM gradient hydrogel. The Ru diffusion gradient equates to a stiffness gradient and models physiology of the scarred skin. Crosslinking in Ru-dECM hydrogels results in a 23-fold increase in stiffness from a stiffness similar to that of normal skin. Collagen fiber density increases in a stiffness-dependent fashion while stress relaxation also alters, with one additional Maxwell element necessary for characterizing Ru-dECM. Alignment of fibroblasts encapsulated in hydrogels suggests that the stiffness gradient directs fibroblasts to orientate at ∼45 ° in regions below 120 kPa. In areas above 120 kPa, fibroblasts decrease the stiffness prior to adjusting their orientation. Furthermore, fibroblasts remodel their surrounding ECM in a gradient-dependent fashion, with rearrangement of cell-surrounding ECM in high-stiffness areas, and formation of interlaced collagen bundles in low-stiffness areas. Overall, this study shows that fibroblasts remodel their local environment to generate an optimal ECM mechanical and topographical environment. STATEMENT OF SIGNIFICANCE: This study developed a versatile in vitro model with a gradient stiffness using skin-derived ECM hydrogel with unchanged biochemical environment. Using Ruthenium crosslinking, a 20-fold stiffness increase was achieved as observed in fibrotic skin. The interaction between fibroblasts and matrix depends on changes in the matrix stiffness. The stiffness gradient directed the alignment of fibroblasts with ∼45° in regions with≤ 120 kPa. The cells in regions with the higher stiffness decreased stiffness first and then oriented themselves. Furthermore, fibroblasts remodeled surrounding ECM and regulated its mechanics in a gradient-dependent fashion to reach an optimal condition. Our study highlights the dynamic interplay between cells and surrounding matrix, shedding light on potential mechanisms and strategies to target scar formation and remodeling.