Extracellular Matrix Expansion Research Articles

610 Myofibroblasts Are Not Characteristic Features of Keloid Lesions

IntroductionKeloids are disfiguring, fibroproliferative lesions that can result from an abnormal wound healing process. Keloids are characterized by excessive and abnormal deposition of extracellular matrix and unrestrained expansion beyond the original wound boundary. Keloids are challenging to treat, with low response rates to current interventions and high recurrence rates. Development of more effective, targeted therapies will require a deeper understanding of the mechanisms driving keloid pathology. It has been widely reported that keloids are characterized by the presence of myofibroblasts, which are specialized, contractile fibroblasts that express alpha-smooth muscle actin (αSMA) and play important roles during wound healing and tissue remodeling. However, the evidence supporting a role for myofibroblasts in keloid development is inconclusive, with conflicting reports in the literature. This complicates development of more effective therapies, as the benefit of interventions targeting myofibroblasts is unclear. The current study evaluated the presence of αSMA+ myofibroblasts in keloids, and analyzed αSMA expression in keloid-derived fibroblasts, to determine whether myofibroblast presence can be considered diagnostic of keloid lesions.MethodsKeloid, hypertrophic scar (HTS), and normal human skin samples were collected with Institutional Review Board approval and primary fibroblast cultures were established. Myofibroblasts in tissue sections were localized by αSMA immunostaining, and αSMA expression in pericytes of blood vessels was distinguished from αSMA+ myofibroblasts by collagen IV immunostaining of vessels. Expression of αSMA mRNA (ACTA2 gene) was measured using quantitative real-time PCR. Statistical analysis via t test was performed using SigmaPlot.ResultsNormal human skin samples did not exhibit αSMA+ myofibroblasts (0/8), but myofibroblasts were identified in 53% of keloids (8/15) and 60% of HTS (3/5) tissue samples. When present in keloids or HTS, the αSMA+ cells were not uniformly distributed, but were often observed in clusters in the deeper regions of the lesions. Comparison of ACTA2 mRNA expression did not reveal any significant differences between normal skin and keloid tissues (N=3/group), or between normal fibroblasts (N=7 donors) and keloid fibroblasts (N=9 donors).ConclusionsThe results suggest that although many keloids contain αSMA+ myofibroblasts, these cells do not appear to be diagnostic for keloids. Therefore, therapies that target myofibroblasts may not be effective for all keloid lesions. However, a limitation of this study is that only excised keloid and HTS tissues, which tend to be larger, more mature lesions, were examined; thus we cannot rule out a potential role for myofibroblasts in early development of abnormal scars.

Pentosan Polysulfate, a Semisynthetic Heparinoid Disease-Modifying Osteoarthritic Drug with Roles in Intervertebral Disc Repair Biology Emulating the Stem Cell Instructive and Tissue Reparative Properties of Heparan Sulfate.

This review highlights the attributes of pentosan polysulfate (PPS) in the promotion of intervertebral disc (IVD) repair processes. PPS has been classified as a disease-modifying osteoarthritic drug (DMOAD) and many studies have demonstrated its positive attributes in the countering of degenerative changes occurring in cartilaginous tissues during the development of osteoarthritis (OA). Degenerative changes in the IVD also involve inflammatory cytokines, degradative proteases, and cell signaling pathways similar to those operative in the development of OA in articular cartilage. PPS acts as a heparan sulfate (HS) mimetic to effect its beneficial effects in cartilage. The IVD contains small cell membrane HS proteoglycans (HSPGs) such as syndecan, and glypican and a large multifunctional HS/chondroitin sulfate (CS) hybrid proteoglycan (HSPG2/perlecan), that have important matrix-stabilizing properties and sequester, control, and present growth factors from the FGF, VEGF, PDGF, and BMP families to cellular receptors to promote cell proliferation, differentiation, and matrix synthesis. HSPG2 also has chondrogenic properties and stimulates the synthesis of extracellular matrix (ECM) components and expansion of cartilaginous rudiments, and has roles in matrix stabilization and repair. Perlecan is a perinuclear and nuclear proteoglycan (PG) in IVD cells with roles in chromatin organization and control of transcription factor activity, immunolocalizes to stem cell niches in cartilage, promotes escape of stem cells from quiescent recycling, differentiation and attainment of pluripotency and migratory properties. These participate in tissue development and morphogenesis, ECM remodeling and repair. PPS also localizes in the nucleus of stromal stem cells, promotes development of chondroprogenitor cell lineages, ECM synthesis and repair and discal repair by resident disc cells. The availability of recombinant perlecan and PPS offers new opportunities in repair biology. These multifunctional agents offer welcome new developments in repair strategies for the IVD.

Cardiac involvement in cystic fibrosis evaluated using cardiopulmonary magnetic resonance

Cystic fibrosis (CF) transmembrane conductance regulator is expressed in myocardium, but cardiac involvement in CF remains poorly understood. The recent development of a combined cardiopulmonary magnetic resonance imaging technology allows for a simultaneous interrogation of cardiac and pulmonary structure and function. The aim of this study was to investigate myocardial manifestations in adults with CF, both in a stable state and during an acute respiratory exacerbation, and to investigate the relationship between cardiac and pulmonary disease. Healthy adult volunteers (n = 12) and adults with CF (n = 10) were studied using a multiparametric cardiopulmonary magnetic resonance protocol. CF patients were scanned during an acute respiratory exacerbation and re-scanned when stable. Stable CF was associated with left ventricular dilatation and hypertrophy (LVH; left ventricular mass: CF 59 ± 9 g/m2 vs. control 50 ± 8 g/m2; p = 0.028). LVH was predominantly driven by extracellular myocardial matrix expansion (extracellular matrix mass: CF 27.5 ± 3.4 g vs. control 23.6 ± 5.2 g; p = 0.006; extracellular volume [ECV]: CF 27.6 [24.7–29.8]% vs. control 24.8 [22.9–26.0]%; p = 0.030). Acute CF was associated with an acute reduction in left ventricular function (ejection fraction: acute 57 ± 3% vs. stable 61 ± 5%; p = 0.025) and there was a suggestion of myocardial oedema. Myocardial oedema severity was strongly associated with the severity of airflow limitation (r = − 0.726, p = 0.017). Multiparametric cardiopulmonary magnetic resonance technology allows for a simultaneous interrogation of cardiac and pulmonary structure and function. Stable CF is associated with adverse myocardial remodelling, including left ventricular systolic dilatation and hypertrophy, driven by myocardial fibrosis. CF exacerbation is associated with acute myocardial contractile dysfunction. There is also a suggestion of myocardial oedema in the acute period which is related to pulmonary disease severity.

Skin Fibrosis and Recovery Is Dependent on Wnt Activation via DPP4

Fibrosis is the life-threatening, excessive accumulation of the extracellular matrix and is sometimes associated with a loss of lipid-filled cells in the skin and other organs. Understanding the mechanisms of fibrosis and associated lipodystrophy and their reversal may reveal new targets for therapeutic intervention. Invivo genetic models are needed to identify key targets that induce recovery from established fibrosis. Wnt signaling is activated in animal and human fibrotic diseases across organs. Here, we developed a genetically inducible and reversible Wnt activation model and showed that it is sufficient to cause fibrotic dermal remodeling, including extracellular matrix expansion and shrinking of dermal adipocytes. Upon withdrawal from Wnt activation, Wnt-induced fibrotic remodeling was reversed in mouse skin-fully restoring skin architecture. Next, we demonstrated CD26/ DPP4 is a Wnt/β-catenin-responsive gene and a functional mediator of fibrotic transformation. We provide genetic evidence that the Wnt/DPP4 axis is required to drive fibrotic dermal remodeling and is associated with human skin fibrosis severity. Remarkably, DPP4 inhibitors can be repurposed to accelerate recovery from established Wnt-induced fibrosis. Collectively, this study identifies Wnt/DPP4 axis as a key driver of extracellular matrix homeostasis and dermal fat loss, providing therapeutic avenues to manipulate the onset and reversal of tissue fibrosis.

Abstract 13878: Activated Fibroblasts and IL-1β Mediate Fibrotic Cardiac Remodeling Observed in Premature Ventricular Contraction-Induced Cardiomyopathy

Introduction: The relevance of fibrosis in premature ventricular contraction-induced cardiomyopathy (PVC-CM) remains unclear. Recent studies suggest that fibrosis promotes adverse extracellular matrix (ECM) remodeling that has been implicated in arrhythmogenesis. However, fibrotic areas and fibroblast activation have not been characterized in PVC-CM. The objective of this study was to assess cardiac remodeling by collagen fiber deposition, fibroblast activation (using αSMA as a marker), and the pro-fibrotic cytokine IL-1β level in LV myocardium. Hypothesis: Activation of fibroblasts promotes adverse cardiac remodeling in PVC-CM. Methods: Modified pacemakers were implanted in canines, half of them developing PVC-CM after 12 weeks of bigeminal PVCs (50% burden at 200-220 ms coupling interval), while the remaining were not exposed to PVCs (sham). Paraffin-embedded or simple formalin-fixed cardiac samples were sectioned and stained with Sirius Red/Fast Green (SR/FG) or immunofluorescence (IF) to assess collagen fiber deposits and αSMA content, respectively. Expression of IL-1β and αSMA were assessed by Western blot. Results: The percentage of fibrosis was three times greater in PVC-CM tissues compared to sham (10.2 ± 0.98 vs 3.3 ± 0.53, p = 0.014), consistent with an increase in αSMA (1 ± 0.08 vs 1.93 ±0.33, p < 0.05) and IL-1β expression (1 ± 0.09 vs 1.69 ± 0.11, p < 0.01). The figure shows differences in collagen fibers with SR/FG and αSMA with IF in (A) sham and (B) PVC-CM. Increased αSMA and IL-1β support the hypothesis that fibroblast activation is a key component in the PVC-CM phenotype. Conclusion: These findings strongly suggest that fibroblast activation during PVCs promote ECM expansion causing persistent structural remodeling which may disrupt electrical signal propagation and potentially contribute to arrhythmogenesis and worsening ventricular function associated with PVC-CM.

Dermal extracellular matrix response to facetem implant: a randomised controlled experimental study

Background. A leading concept in modern regenerative medicine is the perspective of using own body resources to remodel organs and tissues via the formation of “living scaffold”. A pivotal role in the formation of dermal scaffold is played by fibroblasts that produce extracellular matrix (ECM).Aim. A study of the fibroblast activation mechanism mediating synthesis of the dermal ECM fibrillar component under the Facetem filler administration.Methods. The experimental trial was conducted in Wistar male rats (72 animals). Animals had 0.05 mL Facetem (Korea) injections subdermally. The filler is a calcium-containing product featuring gradual degradation delivered through structural microspheric properties of the Lattice-pore technology. Biological material was sampled at weeks 1 and 2 of months 1, 2, 3 and 5. Tissues were paraffin-embedded in standard histological assays and stained with Mallory’s trichrome, Picrosirius red in polarisation microscopy and immunohistochemistry with collagen types I, III and elastin antibodies (Abcam).Results. Collagen distribution in dermis and the filling zone suggests that collagen production occurs by week 2 of the Facetem filler placement followed by an increase in synthesised matrix volume to 4.39 ± 0.7 for collagen type I and 3.9 ± 0.2 for collagen type III (p < 0.05). The synthetic activity of fi broblasts reduces by month 3, albeit with collagen production remaining above control even by the end of month 5. Elastin synthesis also initiates by week 2 of the filler injection in dermis and grows by month 3.Conclusion. The presence of Facetem filler triggers a foreign body inflammatory response in dermis. This multifactorial process initiates with protein adsorption proceeding to dermal cell recruitment and modulation of fibroblasts and macrophages. Activation of these cell types induces neocollagenesis entailing the extracellular matrix synthesis and expansion in dermis.

Collagen-Targeted Peptides for Molecular Imaging of Diffuse Cardiac Fibrosis.

BackgroundCardiac fibrosis is the excessive deposition of extracellular matrix in the heart, triggered by a cardiac insult, aging, genetics, or environmental factors. Molecular imaging of the cardiac extracellular matrix with targeted probes could improve diagnosis and treatment of heart disease. However, although this technology has been used to demonstrate focal scarring arising from myocardial infarction, its capacity to demonstrate extracellular matrix expansion and diffuse cardiac fibrosis has not been assessed.Methods and ResultsHere, we report the use of collagen‐targeted peptides labeled with near‐infrared fluorophores for the detection of diffuse cardiac fibrosis in the β2‐AR (β‐2‐adrenergic receptor) overexpressing mouse model and in ischemic human hearts. Two approaches were evaluated, the first based on a T peptide that binds matrix metalloproteinase‐2‐proteolyzed collagen IV, and the second on the cyclic peptide EP‐3533, which targets collagen I. The systemic and cardiac uptakes of both peptides (intravenously administered) were quantified ex vivo by near‐infrared imaging of whole organs, tissue sections, and heart lysates. The peptide accumulation profiles corresponded to an immunohistochemically‐validated increase in collagen types I and IV in hearts of transgenic mice versus littermate controls. The T peptide could encouragingly demonstrate both the intermediate (7 months old) and severe (11 months old) cardiomyopathic phenotypes. Co‐immunostainings of fluorescent peptides and collagens, as well as reduced collagen binding of a control peptide, confirmed the collagen specificity of the tracers. Qualitative analysis of heart samples from patients with ischemic cardiomyopathy compared with nondiseased donors supported the collagen‐enhancement capabilities of these peptides also in the clinical settings.ConclusionsTogether, these observations demonstrate the feasibility and translation potential of molecular imaging with collagen‐binding peptides for noninvasive imaging of diffuse cardiac fibrosis.

Role and Evolution of the Extracellular Matrix in the Acquisition of Complex Multicellularity in Eukaryotes: A Macroalgal Perspective.

Multicellular eukaryotes are characterized by an expanded extracellular matrix (ECM) with a diversified composition. The ECM is involved in determining tissue texture, screening cells from the outside medium, development, and innate immunity, all of which are essential features in the biology of multicellular eukaryotes. This review addresses the origin and evolution of the ECM, with a focus on multicellular marine algae. We show that in these lineages the expansion of extracellular matrix played a major role in the acquisition of complex multicellularity through its capacity to connect, position, shield, and defend the cells. Multiple innovations were necessary during these evolutionary processes, leading to striking convergences in the structures and functions of the ECMs of algae, animals, and plants.

MO014DANHONG INJECTION INHIBITS LIPOPOLYSACCHARIDE-ENHANCED CELL PROLIFERATION OF RAT RENAL MESANGIAL CELLS VIA NF-ΚB SIGNALING PATHWAY

Abstract Background and Aims Mesangioproliferative glomerulonephritis (MsPGN) is well known as one of the leading causes of end-stage renal failure (ESRD), especially in the Asian population. The principal characteristic of MsPGN is the over proliferation of glomerular mesangial cells (MCs) accompanied by expansion of the extracellular matrix (ECM). Our previous studies demonstrated that Danhong injection (DHI), is a traditional Chinese medicine injection, could improve the renal function and inhibit the MCs proliferation and ECM expansion in rats with MsPGN. However, the molecular mechanisms have not been fully elucidated. To explore the potential mechanisms of DHI in the treatment of MsPGN, we investigated the effects of DHI on lipopolysaccharide (LPS)-induced nuclear factor-kappa B (NF-κB) activation and its downstream inflammatory mediators, such as intercellular adhesion molecule-1 (ICAM-1), transforming growth factor-beta 1 (TGF-β1), inducible nitric oxide synthase (iNOS) and fibronectin (FN) protein expression in rat MCs. Method The rat MCs treated with different concentrations of DHI (0, 50, 100, 200, 500, 1000, and 2000 uL/L) for 12 h, then incubated with or without 100 ng/ml LPS for another 24 h. Subsequently, cell proliferation was determined by Cell Counting Kit-8 (CCK8). Furthermore, the rat MCs treated with low-dose DHI (250 uL/L), median-dose DHI (500 uL/L) and high-dose DHI (1000 uL/L) for 12 h or Ammonium pyrrolidine dithiocarbamate (PDTC) for 30 min before 24h treatment of LPS. Then the activation of NF-κB was detected by Western blot and immunofluorescence. The protein levels of ICAM-1, TGF-β1, iNOS and FN in rat MCs were detected by Western blot. Results The results of CCK-8 revealed that DHI significantly suppressed LPS-induced cell proliferation (shown in Fig.1). LPS stimulation resulted in a significant increment of p65 contents in nucleus and a decrement of p65 contents in cytoplasm in rat MCs compared with NC. PDTC and DHI exerted potent inhibitory effect on increasing expression of p65 in nucleus and decreasing in cytoplasm compared with LPS treatment group. The inhibitory effect on NF-κB nuclear translocation of DHI was in a dose- dependent manner (shown in Fig.2). The Western blot assay showed that the protein level of IκB-α in cytoplasm treated by LPS decreased significantly compared with that in control (shown in Fig. 3) and this decrement was significantly reversed by PDTC and DHI. In addition, the protein expression of ICAM-1, TGF-β1, iNOS and FN was also inhibited by PDTC and DHI (shown in Fig. 4). Conclusion DHI significantly repressed LPS-induced cell proliferation and FN expression in rat MCs through inhibiting the activation of NF-κB signaling pathway and protein expression of its downstream inflammatory mediators. The ameliorative effects of DHI on MsPGN might be associated with this inhibition effect on NF-B signaling pathway.

Calcium‐Dependent Dual Oxidase 2 is a Novel Source of Reactive Oxygen Species Implicated in Glomerular Mesangial Cell Fibrotic Response to Angiotensin II

The vasoactive peptide angiotensin II (Ang II) contributes to the initiation and the progression of glomerular fibrosis via activation of glomerular mesangial cells (MCs) and subsequent extracellular matrix expansion. We have previously shown that oxidative stress is critical for MC fibrotic response to Ang II. Here, we demonstrate that Dual oxidase 2 (Duox2), a member of the Nox/Duox family of NADPH oxidases, is present in MCs and that its protein expression is upregulated by Ang II. Small interfering RNA (siRNA)-mediated downregulation of Duox2 significantly reduces Ang II-induced increase in reactive oxygen (ROS) generation and prevents the stimulatory effect of Ang II on MC fibrotic injury (as assessed by measuring a-smooth muscle actin and fibronectin expression). To demonstrate that Duox2 activation is Ca2+-dependent, we show that the extracellular Ca2+ chelator BAPTA prevented Ang II-induced ROS generation and the stimulation of MC fibrotic injury by Ang II. Moreover, treatment of MCs with ionomycin resulted in increased ROS production and enhanced MC fibrotic injury. These effects were abrogated by siRNA targeting Duox2. Fura-2 fluorescence was utilized as well to show calcium mobilization in response to Ang II and this effect was abrogated with siDuox2 transfection. These data indicate that Ang II-stimulated Duox2 activation and ROS generation subsequently lead to MC fibrotic injury. In summary, we have identified a novel role for Duox2 as a major source of ROS in response to Ang II and established the significance of Duox2 in Ang II-mediated MC activation and fibrotic injury. Therapeutic targeting of this pathway may prevent or reverse pathophysiologic manifestations of renal fibrotic diseases.

612 Wnt signaling induces fibrotic fat loss via DPP4 in skin fibrosis

Fibrotic diseases involve a loss of fat or lipid-filled cells in several organs including the lungs, liver, and skin in addition to expansion of extracellular matrix (ECM). Dermal fibrosis including excess scarring, scleroderma, and eczema, affects about 40 million people, yet there are no therapies for its reversal. Skin is unique among fibrotic organs due to its accessibility and distinct fat compartment, called dermal white adipose tissue (DWAT). Reduced lipid among mature adipocytes which comprise the DWAT has implications for their many functions. The mechanisms governing fibrotic DWAT lipid depletion and the effects of lipid depletion are not known. Wnt signaling is dysregulated among fibrotic tissues and sustained Wnt signaling in mouse dermis is sufficient to cause dermal fibrosis including DWAT lipid depletion through previously unknown mediators. Here we test the following hypothesis: Induced Wnt signaling stimulates lipid breakdown via dipeptidyl peptidase 4 (DPP4) in dermal adipocytes impacting ECM expansion. Using an inducible and reversible genetic mouse model of dermal Wnt activation, we identify cellular mechanisms of lipid depletion and recovery. Wnt activation leads to breakdown of intracellular lipid in DWAT, stimulation of the lipolytic axis, and increased ECM remodeling, all of which recover during reversal. Genetic ablation of candidate Wnt-responsive factor, DPP4, in our model leads to DWAT preservation, attenuated lipid breakdown, diminished ECM remodeling, and reduced dermal thickening. Finally, chemical inhibition of DPP4 in our mouse model leads to accelerated recovery of DWAT and dermis. Together these data demonstrate that Wnt-DPP4 modulates lipid homeostasis in adipocytes, impacting neighboring dermal fibroblasts. Thus, our results suggest that treatment of dermal fibrosis with FDA-approved DPP4 inhibitors may effectively target lipid depletion, a new cellular player in fibrosis.

HDAC Inhibition Reverses Preexisting Diastolic Dysfunction and Blocks Covert Extracellular Matrix Remodeling.

Diastolic dysfunction (DD) is associated with the development of heart failure and contributes to the pathogenesis of other cardiac maladies, including atrial fibrillation. Inhibition of histone deacetylases (HDACs) has been shown to prevent DD by enhancing myofibril relaxation. We addressed the therapeutic potential of HDAC inhibition in a model of established DD with preserved ejection fraction. Four weeks after uninephrectomy and implantation with deoxycorticosterone acetate pellets, when DD was clearly evident, 1 cohort of mice was administered the clinical-stage HDAC inhibitor ITF2357/Givinostat. Echocardiography, blood pressure measurements, and end point invasive hemodynamic analyses were performed. Myofibril mechanics and intact cardiomyocyte relaxation were assessed ex vivo. Cardiac fibrosis was evaluated by picrosirius red staining and second harmonic generation microscopy of left ventricle (LV) sections, RNA sequencing of LV mRNA, mass spectrometry-based evaluation of decellularized LV biopsies, and atomic force microscopy determination of LV stiffness. Mechanistic studies were performed with primary rat and human cardiac fibroblasts. HDAC inhibition normalized DD without lowering blood pressure in this model of systemic hypertension. In contrast to previous models, myofibril relaxation was unimpaired in uninephrectomy/deoxycorticosterone acetate mice. Furthermore, cardiac fibrosis was not evident in any mouse cohort on the basis of picrosirius red staining or second harmonic generation microscopy. However, mass spectrometry revealed induction in the expression of >100 extracellular matrix proteins in LVs of uninephrectomy/deoxycorticosterone acetate mice, which correlated with profound tissue stiffening based on atomic force microscopy. ITF2357/Givinostat treatment blocked extracellular matrix expansion and LV stiffening. The HDAC inhibitor was subsequently shown to suppress cardiac fibroblast activation, at least in part, by blunting recruitment of the profibrotic chromatin reader protein BRD4 (bromodomain-containing protein 4) to key gene regulatory elements. These findings demonstrate the potential of HDAC inhibition as a therapeutic intervention to reverse existing DD and establish blockade of extracellular matrix remodeling as a second mechanism by which HDAC inhibitors improve ventricular filling. Our data reveal the existence of pathophysiologically relevant covert or hidden cardiac fibrosis that is below the limit of detection of histochemical stains such as picrosirius red, highlighting the need to evaluate fibrosis of the heart using diverse methodologies.

Asymmetric Hapln1a drives regionalized cardiac ECM expansion and promotes heart morphogenesis in zebrafish development.

AimsVertebrate heart development requires the complex morphogenesis of a linear tube to form the mature organ, a process essential for correct cardiac form and function, requiring coordination of embryonic laterality, cardiac growth, and regionalized cellular changes. While previous studies have demonstrated broad requirements for extracellular matrix (ECM) components in cardiac morphogenesis, we hypothesized that ECM regionalization may fine tune cardiac shape during heart development.Methods and resultsUsing live in vivo light sheet imaging of zebrafish embryos, we describe a left-sided expansion of the ECM between the myocardium and endocardium prior to the onset of heart looping and chamber ballooning. Analysis using an ECM sensor revealed the cardiac ECM is further regionalized along the atrioventricular axis. Spatial transcriptomic analysis of gene expression in the heart tube identified candidate genes that may drive ECM expansion. This approach identified regionalized expression of hapln1a, encoding an ECM cross-linking protein. Validation of transcriptomic data by in situ hybridization confirmed regionalized hapln1a expression in the heart, with highest levels of expression in the future atrium and on the left side of the tube, overlapping with the observed ECM expansion. Analysis of CRISPR-Cas9-generated hapln1a mutants revealed a reduction in atrial size and reduced chamber ballooning. Loss-of-function analysis demonstrated that ECM expansion is dependent upon Hapln1a, together supporting a role for Hapln1a in regionalized ECM modulation and cardiac morphogenesis. Analysis of hapln1a expression in zebrafish mutants with randomized or absent embryonic left–right asymmetry revealed that laterality cues position hapln1a-expressing cells asymmetrically in the left side of the heart tube.ConclusionWe identify a regionalized ECM expansion in the heart tube which promotes correct heart development, and propose a novel model whereby embryonic laterality cues orient the axis of ECM asymmetry in the heart, suggesting these two pathways interact to promote robust cardiac morphogenesis.

FoxP1 is a transcriptional repressor associated with cancer cachexia that induces skeletal muscle wasting and weakness.

BackgroundSkeletal muscle wasting is a devastating consequence of cancer that affects up to 80% of cancer patients and associates with reduced survival. Herein, we investigated the biological significance of Forkhead box P1 (FoxP1), a transcriptional repressor that we demonstrate is up‐regulated in skeletal muscle in multiple models of cancer cachexia and in cachectic cancer patients.MethodsInducible, skeletal muscle‐specific FoxP1 over‐expressing (FoxP1iSkmTg/Tg) mice were generated through crossing conditional Foxp1a transgenic mice with HSA‐MCM mice that express tamoxifen‐inducible Cre recombinase under control of the skeletal muscle actin promoter. To determine the requirement of FoxP1 for cancer‐induced skeletal muscle wasting, FoxP1‐shRNA was packaged and targeted to muscles using AAV9 delivery prior to inoculation of mice with Colon‐26 Adenocarcinoma (C26) cells.ResultsUp‐regulation of FoxP1 in adult skeletal muscle was sufficient to induce features of cachexia, including 15% reduction in body mass (P < 0.05), and a 16–27% reduction in skeletal muscle mass (P < 0.05) that was characterized by a 20% reduction in muscle fibre cross‐sectional area of type IIX/B muscle fibres (P = 0.020). Skeletal muscles from FoxP1iSkmTg/Tg mice also showed significant damage and myopathy characterized by the presence of centrally nucleated myofibres, extracellular matrix expansion, and were 19–26% weaker than controls (P < 0.05). Transcriptomic analysis revealed FoxP1 as a potent transcriptional repressor of skeletal muscle gene expression, with enrichment of pathways related to skeletal muscle structure and function, growth signalling, and cell quality control. Because FoxP1 functions, at least in part, as a transcriptional repressor through its interaction with histone deacetylase proteins, we treated FoxP1iSkmTg/Tg mice with Trichostatin A, and found that this completely prevented the loss of muscle mass (p = 0.007) and fibre atrophy (P < 0.001) in the tibialis anterior. In the context of cancer, FoxP1 knockdown blocked the cancer‐induced repression of myocyte enhancer factor 2 (MEF2)‐target genes critical to muscle differentiation and repair, improved muscle ultrastructure, and attenuated muscle fibre atrophy by 50% (P < 0.05).ConclusionsIn summary, we identify FoxP1 as a novel repressor of skeletal muscle gene expression that is increased in cancer cachexia, whose up‐regulation is sufficient to induce skeletal muscle wasting and weakness, and required for the normal wasting response to cancer.

PATHOMORPHOLOGICAL CHARACTERISTICS OF IMMUNOCOMPLEX RENAL DISEASE IN PATIENTS WITH IMMUNODEFICIENCY VIRUS AND HEPATITIS C VIRUS, RECEIVING ANTIRETROVIRAL THERAPY

The aim is to verify and describe the morphological substrate of renal impairment in HIV/HCV co-infection among patients receiving ART to assess and predict the morphogenesis of immunocomplex lesions. Materials and methods: To assess and predict the morphogenesis of immunocomplex renal disease, we examined retrospectively the kidney tissue samples of 15 patients, who died with HIV/HCV co-infection and received ART. Histological, histochemical and immunohistochemical research methods were used. Results: Segmental and diffuse mesangial proliferation with extracellular matrix expansion with glomerular damage ≥50% in 9 (60%) cases, and involving &lt;50% of glomeruli in 5 (33%), with CD68 expression as single cells were detected. In 12 (80%) cases, there was uneven swelling and focal proliferation of endothelial cells with the involvement of 20-50% of the glomeruli, as well as the presence of cellular infiltrates in the lumen of capillary loops in 3 (20%) cases with monomorphic intensity in “+”. Sclerotic changes were present in various degrees of severity – from cases of complete glomerulosclerosis with obliteration of the Bowman’s lumen to focal and microfocal depressions 8 (55%), sclerosis 10 (66%), hyalinosis 1 (6%), uneven thickening, focal cleft 8 (55%) and perihilar focal sclerosis. These areas were positive for IgG and C1q complement fractions within the “+”, “++” intensity. Among the study group, no case of HIV-associated nephropathy was found that coincided with the predicted spectrum of kidney damage for patients in this sample. The described morphological changes were mainly verified as immuno-mediated by HCV. Conclusions: A comprehensive morphological study revealed the morphological substrate of kidney damage and its morphogenesis in patients with HIV/HCV co-infection, receiving ART.

Unexpected increase of myocardial extracellular volume fraction in low cardiovascular risk HIV patients

BackgroundPeople living with HIV (PLWH) are prone to develop sub-clinical Cardiovascular (CV) disease, despite the effectiveness of combined Antiretroviral Therapy (cART). Algorithms developed to predict CV risk in the general population could be inaccurate when applied to PLWH. Myocardial Extra-Cellular Matrix (ECM) expansion, measured by computed tomography, has been associated with an increased CV vulnerability in HIV-negative population. Measurement of Myocardial Extra-Cellular Volume (ECV) by computed tomography or magnetic resonance, is considered a useful surrogate for clinical evaluation of ECM expansion. In the present study, we aimed to determine the extent of cardiovascular involvement in asymptomatic HIV-infected patients with the use of a comprehensive cardiac computed tomography (CCT) approach.Materials and methodsIn the present study, ECV in low atherosclerotic CV risk PLWH was compared with ECV of age and gender matched HIV- individuals. 53 asymptomatic HIV + individuals (45 males, age 48 (42.5–48) years) on effective cART (CD4 + cell count: 450 cells/µL (IQR: 328–750); plasma HIV RNA: <37 copies/ml in all subjects) and 18 age and gender matched controls (14 males, age 55 (44.5–56) years) were retrospectively enrolled. All participants underwent CCT protocol to obtain native and postcontrast Hounsfield unit values of blood and myocardium, ECM was calculated accordingly.ResultsThe ECV was significantly higher in HIV + patients than in the control group (ECV: 31% (IQR: 28%-31%) vs. 27.4% (IQR: 25%-28%), p < 0.001). The duration of cART (standardized β = 0.56 (0.33–0.95), p = 0.014) and the years of exposure to HIV infection (standardized β = 0.53 (0.4–0.92), p < 0.001), were positively and strongly associated with ECV values. Differences in ECV (p < 0.001) were also observed regarding the duration of cART exposure (< 5 years, 5–10 years and > 10 years). Moreover, ECV was independently associated with age of participants (standardized β = 0.42 (0.33–0.89), p = 0.084).ConclusionsHIV infection and exposure to antiretrovirals play a detrimental role on ECV expansion. An increase in ECV indicates ECM expansion, which has been associated to a higher CV risk in the general population. The non-invasive evaluation of ECM trough ECV could represent an important tool to further understand the relationship between HIV infection, cardiac pathophysiology and the increased CV risk observed in PLWH.

Abstract 16060: Cardiac Magnetic Resonance Imaging Diastolic Indices Correlate With Cardiac Catheterization Filling Pressures in Pediatric Heart Transplant Recipients

Background: Atrial and ventricular filling pressures are routinely used in pediatric heart transplant (HTx) recipients to assess graft function. Cardiac magnetic resonance (CMR) can provide a non-invasive assessment of diastolic function, but data are lacking in pediatric HTx. We hypothesized that CMR indices of diastolic function correlate with filling pressures, providing a non-invasive approach to assessment of ventricular dysfunction. Method: Pediatric HTx recipients were prospectively enrolled at the time of cardiac catheterization. Pulmonary capillary wedge pressure (PCWP) and right atrial pressure (RAP) were measured. CMR included standard volumetric analysis and T1 and extracellular volume (ECV) mapping in the short axis at the mid-left ventricle (LV) to evaluate extracellular matrix expansion. Filling curves were calculated by contouring every phase in the short axis stack and global longitudinal and circumferential strain (GLS, GCS) were calculated using feature tracking. Correlations were analyzed using Spearman’s correlation. Results: A total of 31 patients with a mean age of 16.2 y/o were included, 9 with biopsy-proven acute rejection. The median time from transplant was 6.9 years and the mean LVEF was 58%. Peak LV ejection rate/end-diastolic volume (PER/EDV) correlated with PCWP (rho=-0.43, p=0.02, Fig 1) and RAP (rho=-0.40, p=0.03). GLS and GCS correlated with RAP (rho=0.51 p=0.007, and rho=0.38 p=0.05, respectively); GLS also correlated with PCWP (rho=0.46, p=0.02). Native T1 time-correlated with PCWP (rho=0.54, p=0.003), while the correlation between ECV and PCWP did not reach statistical significance (rho=0.37, p=0.06). Conclusion: CMR can be a useful non-invasive modality to assess for early signs of diastolic dysfunction in patients after pediatric HTx. Further research is needed to describe the clinical significance of these associations.

Aldosterone-Related Myocardial Extracellular Matrix Expansion in Hypertension in Humans: A Proof-of-Concept Study by Cardiac Magnetic Resonance

ObjectivesThis study sought to assess the respective effects of aldosterone and blood pressure (BP) levels on myocardial fibrosis in humans. BackgroundExperimentally, aldosterone promotes left ventricular (LV) hypertrophy, and interstitial myocardial fibrosis in the presence of high salt intake. MethodsThe study included 20 patients with primary aldosteronism (PA) (high aldosterone and high BP), 20 patients with essential hypertension (HTN) (average aldosterone and high BP), 20 patients with secondary aldosteronism due to Bartter/Gitelman (BG) syndrome (high aldosterone and normal BP), and 20 healthy subjects (HS) (normal aldosterone and normal BP). Participants in each group were of similar age and sex distributions, and asymptomatic. Cardiac magnetic resonance including cine and T1 mapping was performed blind to the study group to quantify global LV mass index, as well as intracellular mass index and extracellular mass index considered as a measure of myocardial fibrosis in vivo. ResultsMedian plasma aldosterone concentration was as follows: PA = 709 pmol/l (interquartile range [IQR]: 430 to 918 pmol/l); HTN = 197 pmol/l (IQR: 121 to 345 pmol/l); BG = 297 pmol/l (IQR: 180 to 428 pmol/l); and HS = 105 pmol/l (IQR: 85 to 227 pmol/l). Systolic BP was as follows: PA = 147 ± 15 mm Hg; HTN = 133 ± 19 mm Hg; BG = 116 ± 9 mm Hg; and HS = 117 ± 12 mm Hg. LV end-diastolic volume showed underloading in BG and overloading in patients with PA (63 ± 13 ml/m2 vs. 82 ± 15 ml/m2; p < 0.0001). Intracellular mass index increased with BP across groups (BG: 36 [IQR: 29 to 41]; HS: 40 [IQR: 36 to 46]; HTN: 51 [IQR: 42 to 54]; PA: 50 [IQR: 46 to 67]; p < 0.0001). Extracellular mass index was similar in BG, HS, and HTN (16 [IQR: 12 to 20]; 15 [IQR: 11 to 18]; and 14 [IQR: 12 to 17], respectively) but 30% higher in PA (21 [IQR: 18 to 29]; p < 0.0001) remaining significant after adjustment for mean BP. ConclusionsOnly primary pathological aldosterone excess combined with high BP increased both extracellular myocardial matrix and intracellular mass. Secondary aldosterone excess with normal BP did not affect extracellular myocardial matrix. (Study of Myocardial Interstitial Fibrosis in Hyperaldosteronism; NCT02938910).

Single-cell RNA sequencing reveals profibrotic roles of distinct epithelial and mesenchymal lineages in pulmonary fibrosis.

Pulmonary fibrosis (PF) is a form of chronic lung disease characterized by pathologic epithelial remodeling and accumulation of extracellular matrix (ECM). To comprehensively define the cell types, mechanisms, and mediators driving fibrotic remodeling in lungs with PF, we performed single-cell RNA sequencing of single-cell suspensions from 10 nonfibrotic control and 20 PF lungs. Analysis of 114,396 cells identified 31 distinct cell subsets/states. We report that a remarkable shift in epithelial cell phenotypes occurs in the peripheral lung in PF and identify several previously unrecognized epithelial cell phenotypes, including a KRT5- /KRT17 + pathologic, ECM-producing epithelial cell population that was highly enriched in PF lungs. Multiple fibroblast subtypes were observed to contribute to ECM expansion in a spatially discrete manner. Together, these data provide high-resolution insights into the complexity and plasticity of the distal lung epithelium in human disease and indicate a diversity of epithelial and mesenchymal cells contribute to pathologic lung fibrosis.

Evolutionary expansion of apical extracellular matrix is required for the elongation of cells in a novel structure.

One of the fundamental gaps in our knowledge of how novel anatomical structures evolve is understanding the origins of the morphogenetic processes that form these features. Here, we traced the cellular development of a recently evolved morphological novelty, the posterior lobe of D. melanogaster. We found that this genital outgrowth forms through extreme increases in epithelial cell height. By examining the apical extracellular matrix (aECM), we also uncovered a vast matrix associated with the developing genitalia of lobed and non-lobed species. Expression of the aECM protein Dumpy is spatially expanded in lobe-forming species, connecting the posterior lobe to the ancestrally derived aECM network. Further analysis demonstrated that Dumpy attachments are necessary for cell height increases during posterior lobe development. We propose that the aECM presents a rich reservoir for generating morphological novelty and highlights a yet unseen role for aECM in regulating extreme cell height.