Increased Extracellular Matrix Protein Research Articles

Perlin noise generation of physiologically realistic cardiac fibrosis

Fibrosis, a pathological increase in extracellular matrix proteins, is a significant health issue that hinders the function of many organs in the body, in some cases fatally. In the heart, fibrosis impacts on electrical propagation in a complex and poorly predictable fashion, potentially serving as a substrate for dangerous arrhythmias. Individual risk depends on the spatial manifestation of fibrotic tissue, and learning the spatial arrangement on the fine scale in order to predict these impacts still relies upon invasive ex vivo procedures. As a result, the effects of spatial variability on the symptomatic impact of cardiac fibrosis remain poorly understood. In this work, we address the issue of availability of such imaging data via a computational methodology for generating new realisations of cardiac fibrosis microstructure. Using the Perlin noise technique from computer graphics, together with an automated calibration process that requires only a single training image, we demonstrate successful capture of collagen texturing in four types of fibrosis microstructure observed in histological sections. We then use this generator to quantitatively analyse the conductive properties of these different types of cardiac fibrosis, as well as produce three-dimensional realisations of histologically-observed patterning. Owing to the generator’s flexibility and automated calibration process, we also anticipate that it might be useful in producing additional realisations of other physiological structures.

Obesity modulates the cellular and molecular microenvironment in the peritoneal cavity: implication for ovarian cancer risk.

Abdominal obesity increases the risk of developing ovarian cancer but the molecular mechanisms of how obesity supports ovarian cancer development remain unknown. Here we investigated the impact of obesity on the immune cell and gene expression profiles of distinct abdominal tissues, focusing on the peritoneal serous fluid (PSF) and the omental fat band (OFB) as critical determinants for the dissemination of ovarian metastases and early metastatic events within the peritoneal cavity. Female C57BL/6 mice were fed a low-fat (LFD) or a high-fat diet (HFD) for 12 weeks until the body weights in the HFD group were significantly higher and the mice displayed an impaired glucose tolerance. Then the mice were injected with the murine ovarian cancer cells (MOSE-LTICv) while remaining on their diets. After 21 days, the mice were sacrificed, tumor burden was evaluated and tissues were harvested. The immune cell composition of abdominal tissues and changes in gene expression in the PSF and OFB were evaluated by flow cytometry and qPCR RT2-profiler PCR arrays and confirmed by qRT-PCR, respectively. Other peritoneal adipose tissues including parametrial and retroperitoneal white adipose tissues as well as blood were also investigated. While limited effects were observed in the other peritoneal adipose tissues, feeding mice the HFD led to distinct changes in the immune cell composition in the PSF and the OFB: a depletion of B cells but an increase in myeloid-derived suppressor cells (MDSC) and mono/granulocytes, generating pro-inflammatory environments with increased expression of cyto- and chemokines, and genes supporting adhesion, survival, and growth, as well as suppression of apoptosis. This was associated with a higher peritoneal tumor burden compared to mice fed a LFD. Changes in cellular and genetic profiles were often exacerbated by the HFD. There was a large overlap in genes that were modulated by both the HFD and the cancer cells, suggesting that this 'genetic fingerprint' is important for ovarian metastases to the OFB. In accordance with the 'seed and soil' theory, our studies show that obesity contributes to the generation of a pro-inflammatory peritoneal environment that supports the survival of disseminating ovarian cancer cells in the PSF and the OFB and enhances the early metastatic adhesion events in the OFB through an increase in extracellular matrix proteins and modulators such as fibronectin 1 and collagen I expression as well as in genes supporting growth and invasion such as Tenacin C. The identified genes could potentially be used as targets for prevention strategies to lower the ovarian cancer risk in women with obesity.

Paxillin regulates liver fibrosis via actin polymerization and ERK activation in hepatic stellate cells.

Liver injury leads to fibrosis and cirrhosis. The primary mechanism underlying the fibrogenic response is the activation of hepatic stellate cells (HSCs), which are 'quiescent' in normal liver but become 'activated' after injury by transdifferentiating into extracellular matrix (ECM)-secreting myofibroblasts. Given that integrins are important in HSC activation and fibrogenesis, we hypothesized that paxillin, a key downstream effector in integrin signaling, might be critical in the fibrosis pathway. Using a cell-culture-based model of HSC activation and in vivo models of liver injury, we found that paxillin is upregulated in activated HSCs and fibrotic livers. Overexpression of paxillin (both in vitro and in vivo) led to increased ECM protein expression, and depletion of paxillin in a novel conditional mouse injury model reduced fibrosis. The mechanism by which paxillin mediated this effect appeared to be through the actin cytoskeleton, which signals to the ERK pathway and induces ECM protein production. These data highlight a novel role for paxillin in HSC biology and fibrosis.

Three-Dimensional Biomaterials with Spatiotemporal Control for Regenerative Tissue Engineering.

At Morehouse College, one of the nation's top liberal arts historically black colleges and universities (HBCU) for African American men, research experiences are used to enhance the liberal arts educational experience. Securing research funding to train HBCU students is highly competitive and challenging due to the review process that is typically vetted by scientists from research-intensive universities who may not be familiar with the HBCU enterprise that may be comprised of insolvent infrastructures. In this Account, the synthesis and preparation of synthetic polymeric biomaterials that are used to facilitate or support changes in biological processes, enhance mechanical properties, and foster tissue growth in three dimensions (3D) under disease conditions will be discussed. The use of biomaterials to help control biological processes in disease states is limited. Hence, the fabrication of 3D scaffolds with chemical variability to grow or repair damaged tissues by inhibiting molecular pathways shows promise by controlling the cellular response to recapitulate 3D tissues and organs. The Mendenhall laboratory at Morehouse College uses 3D biomaterials to solve biological problems by probing cellular mechanistic pathways using natural products and nanoparticles. Toward this end, we have fabricated and manufactured 3D biomaterial scaffolds using chemical strategies to mitigate biological processes to help restore pristine tissue properties. Hydrogels are 3D polymeric matrixes that swell in aqueous environments and support cell growth that later infuriates the 3D matrix to create new tissue(s). In contrast, electrospun fibers use high electric fields to create porous 3D polymeric structures that can be used to create 3D tissue molds.The synergistic use of 3D biomaterial templates that can inhibit cellular damage while providing a mechanically strong scaffold to support regenerative tissue growth is essential to creating the next generation of biomaterials. This approach will require foresight using tools from synthetic biology, molecular biology, autonomous processes, advanced biomanufacturing, and machine learning (ML). The use of several biomaterials has been explored by the Mendenhall laboratory to design, prepare, fabricate, characterize, and evaluate 3D electrospun fibers and hydrogels containing hybrid compositions of polylactic acid (PLA), poly(n-vinylcaprolactam) (PVCL), cellulose acetate (CA), and methacrylated hyaluronic acid (meHA). This work contributed to the newly fabricated PVCL-CA fibers with morphological changes and nanoscale fiber hydrophobic surface properties. While the use of electrospun fibers can create hierarchal scaffolds for bone tissue engineering, the use of injectable gels for nonporous tissues such as articular cartilage presents another compelling biomaterial challenge. Using graft polymerization, we prepared PVLC-graft-HA and studied the effect of lower critical solution temperatures (LCSTs), gelation temperatures, and mechanical properties using temperature-controlled rheology. Additionally, we reported that articular cartilage (chondrocyte) cells seeded in PVCL-g-HA gels and incubated at hypoxia 1% O2 produced a 10-fold increase in extracellular matrix proteins (collagen) after 10 days. This work supported exploring new approaches to protecting chondrocyte cells under hypoxia using a 3D scaffold technology.

Multiscale analysis of human tissue engineered matrices for next generation heart valve applications.

Human tissue-engineered matrices (hTEMs) have been proposed as a promising approach for in situ tissue engineered heart valves (TEHVs). However, there is still a limited understanding on how ECM composition in hTEMs develops over tissue culture time. Therefore, we performed a longitudinal hTEM assessment by 1) multiscale evaluation of hTEM composition during culture time (2, 4, 6-weeks), using (immuno)histology, biochemical assays, and mass spectrometry (LC-MS/MS); 2) analysis of protein pathways involved in ECM development using gene set enrichment analysis (GSEA); and 3) assessment of hTEM mechanical characterization using uniaxial tensile testing. Finally, as a proof-of-concept, TEHVs manufactured using 6-weeks hTEM samples were tested in a pulse duplicator. LC-MS/MS confirmed the tissue culture time-dependent increase in ECM proteins observed in histology and biochemical assays, revealing the most abundant collagens (COL6, COL12), proteoglycans (HSPG2, VCAN), and glycoproteins (FN, TNC). GSEA identified the most represented protein pathways in the hTEM at 2-weeks (mRNA metabolic processes), 4-weeks (ECM production), and 6-weeks (ECM organization and maturation). Uniaxial mechanical testing showed increased stiffness and stress at failure, and reduction in strain over tissue culture time. hTEM-based TEHVs demonstrated promising in vitro performance at both pulmonary and aortic pressure conditions, with symmetric leaflet coaptation and no stenosis. In conclusion, ECM protein abundance and maturation increased over tissue culture time, with consequent improvement of hTEM mechanical characteristics. These findings suggest that longer tissue culture impacts tissue organization, leading to an hTEM that may be suitable for high-pressure applications. STATEMENT OF SIGNIFICANCE: It is believed that the composition of the extracellular matrix (ECM) in the human tissue engineered matrices (hTEM) may favor tissue engineered heart valve (TEHV) remodeling upon implantation. However, the exact protein composition of the hTEM, and how this impacts tissue mechanical properties, remains unclear. Hence, we developed a reproducible rotation-based tissue culture method to produce hTEM samples. We performed a longitudinal assessment using different analytical techniques and mass spectrometry. Our data provided an in-depth characterization of the hTEM proteome with focus on ECM components, their development, and how they may impact the mechanical properties. Based on these results, we manufactured functional hTEM-based TEHVs at aortic-like condition in vitro. These outcomes pose an important step in translating hTEM-based TEHVs into clinics and in predicting their remodeling potential upon implantation.

Cell surface GRP78 regulates TGFβ1-mediated profibrotic responses via TSP1 in diabetic kidney disease.

Introduction: Diabetic kidney disease (DKD) is the leading cause of kidney failure in North America, characterized by glomerular accumulation of extracellular matrix (ECM) proteins. High glucose (HG) induction of glomerular mesangial cell (MC) profibrotic responses plays a central role in its pathogenesis. We previously showed that the endoplasmic reticulum resident GRP78 translocates to the cell surface in response to HG, where it mediates Akt activation and downstream profibrotic responses in MC. Transforming growth factor β1 (TGFβ1) is recognized as a central mediator of HG-induced profibrotic responses, but whether its activation is regulated by cell surface GRP78 (csGRP78) is unknown. TGFβ1 is stored in the ECM in a latent form, requiring release for biological activity. The matrix glycoprotein thrombospondin 1 (TSP1), known to be increased in DKD and by HG in MC, is an important factor in TGFβ1 activation. Here we determined whether csGRP78 regulates TSP1 expression and thereby TGFβ1 activation by HG. Methods: Primary mouse MC were used. TSP1 and TGFβ1 were assessed using standard molecular biology techniques. Inhibitors of csGRP78 were: 1) vaspin, 2) the C-terminal targeting antibody C38, 3) siRNA downregulation of its transport co-chaperone MTJ-1 to prevent GRP78 translocation to the cell surface, and 4) prevention of csGRP78 activation by its ligand, active α2-macroglobulin (α2M*), with the neutralizing antibody Fα2M or an inhibitory peptide. Results: TSP1 transcript and promoter activity were increased by HG, as were cellular and ECM TSP1, and these required PI3K/Akt activity. Inhibition of csGRP78 prevented HG-induced TSP1 upregulation and deposition into the ECM. The HG-induced increase in active TGFβ1 in the medium was also inhibited, which was associated with reduced intracellular Smad3 activation and signaling. Overexpression of csGRP78 increased TSP-1, and this was further augmented in HG. Discussion: These data support an important role for csGRP78 in regulating HG-induced TSP1 transcriptional induction via PI3K/Akt signaling. Functionally, this enables TGFβ1 activation in response to HG, with consequent increase in ECM proteins. Means of inhibiting csGRP78 signaling represent a novel approach to preventing fibrosis in DKD.

The Role of Scleraxis in Inducing Vascular Stiffness

Vascular fibrosis (VF) is one of the most significant pathologies related to blood vessel dysfunction. VF results in an impairment of vascular tone due to an increase in extracellular matrix proteins (ECM) and vascular smooth muscle synthesis within the vessel wall, thus increasing vascular stiffness and reducing lumen diameter. VF is both caused by and exacerbates numerous vascular diseases including diabetes, atherosclerosis and in particular, hypertension, which accounts for 13% of deaths globally (WHO World Health Report, 2002). Multiple studies have investigated various therapeutic approaches to interfere with VF, but to date this condition remains untreated, thus novel approaches to target fibrosis development and progression are urgently required.Our lab identified the transcription factor scleraxis as a novel master regulator of cellular phenotype conversion. We demonstrated that scleraxis is sufficient to induce fibroblast to myofibroblast phenotype conversion, a critical step in the development of fibrosis in many tissue types. Scleraxis also induces Epithelial to Mesenchymal Transition (EMT) and cell migration as part of development and cell differentiation. Some studies have shown that mechanical stretch in tendons increases expression of scleraxis, while additional studies reported that stretch in rabbit aorta induces the vascular smooth muscle proliferative phenotype. Our preliminary data has revealed that scleraxis is expressed in VSMCs in the arterial wall, and we have found elevated scleraxis expression in high pressure versus low pressure regions of vessels. We thus hypothesize that scleraxis is sufficient and necessary to induce vascular fibrosis.Our data shows that scleraxis overexpression increases thickness of the aortic vascular wall in scleraxis overexpression mice, via histological sections, thus contributing to the progression of vascular fibrosis. In vitro studies show that scleraxis overexpression in Human Aortic Smooth Muscle Cells (HAOSMC) induces their proliferation and migration using different assays (CCk8/Edu+ proliferation assay, Transwell migration assay, gene expression assays). Scleraxis overexpression also induces hypertrophy of HAOSMC shown via immunostaining. This suggests that scleraxis overexpression induces VSMC plasticity from contractile to synthetic‐proliferative phenotype, thus inducing VSMC hyperplasia.New & Noteworthy: Our findings indicate that scleraxis plays a role in VSMC plasticity via scleraxis‐ mediated transcriptional regulation of VSMC proliferation and migration. This work has potential relevance to many related diseases that feature VSMC activation, including atherosclerosis and restenosis.

CCN2/CTGF-A Modulator of the Optic Nerve Head Astrocyte.

In primary open-angle glaucoma (POAG), a neurodegenerative disease of the optic nerve (ON) and leading cause of blindness, the optic nerve head (ONH) undergoes marked structural extracellular matrix (ECM) changes, which contribute to its permanent deformation and to degeneration of ON axons. The remodeling process of the ECM causes changes in the biomechanical properties of the ONH and the peripapillary sclera, which is accompanied by an increased reactivity of the resident astrocytes. The molecular factors involved in the remodeling process belong to the Transforming growth factor (TGF)-β superfamily, especially TGF-β2. In previous publications we showed that TGF-β2 induced ECM alterations are mediated by Cellular Communication Network Factor (CCN)2/Connective Tissue Growth Factor (CTGF) and recently we showed that CCN2/CTGF is expressed by astrocytes of the ON under normal conditions. In this study we wanted to get a better understanding of the function of CCN2/CTGF under normal and pathologic conditions. To this end, we analyzed the glial lamina and peripapillary sclera of CCN2/CTGF overexpressing mice and studied the effect of CCN2/CTGF and increasing substratum stiffness on murine ON astrocytes in vitro. We observed enhanced astrocyte reactivity in the ONH, increased ECM protein synthesis in the peripapillary sclera and increased Ccn2/Ctgf expression in the ONH during the pathologic development in situ. CCN2/CTGF treatment of primary murine ON astrocytes induced a higher migration rate, and increase of ECM proteins including fibronectin, elastin and collagen type III. Furthermore, the astrocytes responded to stiffer substratum with increased glial fibrillary acidic protein, vimentin, actin and CCN2/CTGF synthesis. Finally, we observed the reinforced appearance of CCN2/CTGF in the lamina cribrosa of glaucomatous patients. We conclude that reactive changes in ONH astrocytes, induced by the altered biomechanical characteristics of the region, give rise to a self-amplifying process that includes increased TGF-β2/CCN2/CTGF signaling and leads to the synthesis of ECM molecules and cytoskeleton proteins, a process that in turn augments the stiffness at the ONH. Such a scenario may finally result in a vicious circle in the pathogenesis of POAG. The transgenic CTGF-overexpressing mouse model might be an optimal model to study the chronic pathological POAG changes in the ONH.

The Th17/IL-17 Axis and Kidney Diseases, With Focus on Lupus Nephritis.

Systemic lupus erythematosus (SLE) is a disease characterized by dysregulation and hyperreactivity of the immune response at various levels, including hyperactivation of effector cell subtypes, autoantibodies production, immune complex formation, and deposition in tissues. The consequences of hyperreactivity to the self are systemic and local inflammation and tissue damage in multiple organs. Lupus nephritis (LN) is one of the most worrying manifestations of SLE, and most patients have this involvement at some point in the course of the disease. Among the effector cells involved, the Th17, a subtype of T helper cells (CD4+), has shown significant hyperactivation and participates in kidney damage and many other organs. Th17 cells have IL-17A and IL-17F as main cytokines with receptors expressed in most renal cells, being involved in the activation of many proinflammatory and profibrotic pathways. The Th17/IL-17 axis promotes and maintains repetitive tissue damage and maladaptive repair; leading to fibrosis, loss of organ architecture and function. In the podocytes, the Th17/IL-17 axis effects include changes of the cytoskeleton with increased motility, decreased expression of health proteins, increased oxidative stress, and activation of the inflammasome and caspases resulting in podocytes apoptosis. In renal tubular epithelial cells, the Th17/IL-17 axis promotes the activation of profibrotic pathways such as increased TGF-β expression and epithelial-mesenchymal transition (EMT) with consequent increase of extracellular matrix proteins. In addition, the IL-17 promotes a proinflammatory environment by stimulating the synthesis of inflammatory cytokines by intrinsic renal cells and immune cells, and the synthesis of growth factors and chemokines, which together result in granulopoiesis/myelopoiesis, and further recruitment of immune cells to the kidney. The purpose of this work is to present the prognostic and immunopathologic role of the Th17/IL-17 axis in Kidney diseases, with a special focus on LN, including its exploration as a potential immunotherapeutic target in this complication.

Lumbar and ventricular CSF concentrations of extracellular matrix proteins before and after shunt surgery in idiopathic normal pressure hydrocephalus

BackgroundIdiopathic normal pressure hydrocephalus (iNPH) is a reversible CNS disease characterized by disturbed cerebrospinal fluid (CSF) dynamics. Changes in the extracellular matrix (ECM) composition might be involved in the pathophysiology of iNPH. The aim of this study was to explore possible differences between lumbar and ventricular CSF concentrations of the ECM markers brevican and neurocan, matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-1 (TIMP-1) and their relation to clinical symptoms in iNPH patients before and after shunt surgery.MethodsPaired lumbar and ventricular CSF was collected from 31 iNPH patients, before and four months after shunt surgery. CSF was analysed for concentrations of tryptic peptides originating from brevican and neurocan using a mass spectrometry-based panel, and for MMP-1, -2, -9, -10 and TIMP-1 using fluorescent or electrochemiluminescent immunoassays.ResultsBrevican and neurocan peptide levels were not influenced by CSF origin, but MMP-1, -2, -10 and TIMP-1 were increased (p ≤ 0.0005), and MMP-9 decreased (p ≤ 0.0003) in lumbar CSF compared with ventricular CSF. There was a general trend of ECM proteins to increase following shunt surgery. Ventricular TIMP-1 was inversely correlated with overall symptoms (rho = − 0.62, p < 0.0001). CSF concentrations of the majority of brevican and neurocan peptides were increased in iNPH patients with a history of cardiovascular disease (p ≤ 0.001, AUC = 0.84–0.94) compared with those without.ConclusionLevels of the CNS-specific proteins brevican and neurocan did not differ between the lumbar and ventricular CSF, whereas the increase of several CNS-unspecific MMPs and TIMP-1 in lumbar CSF suggests contribution from peripheral tissues. The increase of ECM proteins in CSF following shunt surgery could indicate disturbed ECM dynamics in iNPH that are restored by restitution of CSF dynamics.

Abstract 3958: Establishment of an in vitro model to study cancer associated fibroblasts and their direct effect on desmoplasia

Abstract Background: The extracellular matrix (ECM) and desmoplasia is emerging as key components in defining prognosis and response to treatment. Cancer associated fibroblasts (CAFs) are vital contributors to the desmoplastic reaction and novel ambitious therapies targeting tumor specific ECM and CAFs are being developed. However, it is still debated whether in fact desmoplasia acts as a barrier to- or promoter of - tumor growth. Therefore, there is a need to establish novel models of desmoplasia to improve the understanding of CAFs and associated cancer specific ECM alterations in order to be successful in drug development and prediction of treatment success. By applying the “Scar-in-a-jar” (SiaJ) model, we established an in vitro model including healthy fibroblasts and CAFs derived from the pancreas to study ECM changes and desmoplasia. Methods: Primary healthy fibroblasts and CAFs were cultured for up to 12 days in the presence or absence of TGF-β. Type I (PRO-C1), type III (PRO-C3) and type VI (PRO-C6) collagen formation were quantified in cell supernatant using enzyme-linked immunosorbent assays. Metabolic activity was evaluated by AlamarBlue. Results: In healthy fibroblasts no collagen formation could be detected without TGF-β. However, the presence of TGF-β induced PRO-C1, PRO-C3 and PRO-C6 formation with levels increasing up to 20-fold (p&amp;lt;0.0001). Furthermore, PRO-C1, PRO-C3 and PRO-C6 levels where significantly increased in CAFs compared to healthy fibroblasts independent of TGF-β stimulation. There was no difference in metabolic activity across cell line. Conclusions: TGF-β stimulation led to increased ECM protein expression in healthy fibroblasts but not in CAFs. However, CAFs expressed higher levels of collagens than healthy fibroblasts independent of TGF-β. These results suggest a distinct role of CAFs in their contribution to desmoplasia compared to healthy fibroblasts. The “Scar-in-a-Jar” model can provide a novel in vitro tool to study CAF activity and their direct effect on desmoplasia. Citation Format: Neel Ingemann Nissen, Morten Karsdal, Nicholas Willumsen. Establishment of an in vitro model to study cancer associated fibroblasts and their direct effect on desmoplasia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3958.

Cardiac α-Actin (ACTC1) Gene Mutation Causes Atrial-Septal Defects Associated With Late-Onset Dilated Cardiomyopathy.

Familial atrial septal defect (ASD) has previously been attributed primarily to mutations in cardiac transcription factors. Here, we report a large, multi-generational family (78 members) with ASD combined with a late-onset dilated cardiomyopathy and further characterize the consequences of mutant α-actin. We combined a genome-wide linkage analysis with cell biology, microscopy, and molecular biology tools to characterize a novel ACTC1 (cardiac α-actin) mutation identified in association with ASD and late-onset dilated cardiomyopathy in a large, multi-generational family. Using a genome-wide linkage analysis, the ASD disease locus was mapped to chromosome 15q14 harboring the ACTC1 gene. In 15 affected family members, a heterozygous, nonsynonymous, and fully penetrant mutation (p. Gly247Asp) was identified in exon 5 of ACTC1 that was absent in all healthy family members (n=63). In silico tools predicted deleterious consequences of this variant that was found absent in control databases. Ultrastructural analysis of myocardial tissue of one of the mutation carriers showed sarcomeric disarray, myofibrillar degeneration, and increased apoptosis, while cardiac proteomics revealed a significant increase in extracellular matrix proteins. Consistently, structural defects and increased apoptosis were also observed in neonatal rat ventricular cardiomyocytes overexpressing the mutant, but not native human ACTC1. Molecular dynamics studies and additional mechanistic analyses in cardiomyocytes confirmed actin polymerization/turnover defects, thereby affecting contractility. A combined phenotype of ASD and late-onset heart failure was caused by a heterozygous, nonsynonymous ACTC1 mutation. Mechanistically, we found a shared molecular mechanism of defective actin signaling and polymerization in both cardiac development and contractile function. Detection of ACTC1 mutations in patients with ASD may thus have further clinical implications with regard to monitoring for (late-onset) dilated cardiomyopathy.

6-Phosphogluconate Dehydrogenase Links Cytosolic Carbohydrate Metabolism to Protein Secretion via Modulation of Glutathione Levels

The proteinaceous extracellular matrix (ECM) is vital for the survival, proliferation, migration, and differentiation of many types of cancer. However, little is known regarding metabolic pathways required for ECM secretion. By using an unbiased computational approach, we searched for enzymes whose suppression may lead to disruptions in protein secretion. Here, we show that 6-phosphogluconate dehydrogenase (PGD), a cytosolic enzyme involved in carbohydrate metabolism, is required for ER structural integrity and protein secretion. Chemical inhibition or genetic suppression of PGD activity led to cell stress accompanied by significantly expanded ER volume and was rescued by compensating endogenous glutathione supplies. Our results also suggest that this characteristic ER-dilation phenotype may be a general marker indicating increased ECM protein congestion inside cells and decreased secretion. Thus, PGD serves as a link between cytosolic carbohydrate metabolism and protein secretion.

Proteomic Analysis Identifies Tenascin-C Expression Is Upregulated in Uterine Fibroids.

Uterine leiomyomas (fibroids) are the most common gynecological tumors, which are enriched in the extracellular matrix (ECM). Fibroids are leading cause of abnormal uterine bleeding and hysterectomy. One of the major questions yet to be answered is the overproduction of specific ECM components in human uterine fibroids, particularly in relation to mutations in the driver gene mediator complex subunit 12 ( MED12). Surgical specimens from 14 patients with uterine leiomyoma having fibroids and corresponding adjacent normal myometrium (ANM) were utilized to analyze genetic and proteomic expression patterns in the tissue samples. MED12 mutations in the fibroids were screened by Sanger sequencing. iTRAQ was used to label the peptides in small-, medium-, and large-sized fibroid samples of annotated MED12 mutation from the same patient. The mixtures of the peptides were fractionated by hydrophilic interaction liquid chromatography (HILIC) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the differential expression proteins. Using isobaric tagged-based quantitative mass spectrometry on 3 selected patients, ECM-related protein tenascin-C (TNC) was observed significantly upregulated (>1.5-fold) with a confidence corresponding to false discovery rate (FDR) <1% in small-, medium-, and large-sized fibroid samples regardless of MED12 mutation status. The TNC was validated on additional patient samples using Western blotting (WB) and immunohistochemistry (IHC) and confirmed significant overexpression of this protein in fibroids compared to matched ANM. Proteomic analyses have identified the increased ECM protein expression, TNC, as a hallmark of uterine fibroids regardless of MED12 mutations. Further functional studies focusing on the upregulated ECM proteins in leiomyogenesis will lead to the identification of novel ECM drug targets for fibroid treatment.

Meprin β deficiency associated with increased ECM protein buildup and reduced macrophage infiltration in mouse kidneys subjected to Unilateral Ureteral Obstruction (UUO)

Meprins are zinc dependent metalloproteases that are abundantly expressed in the brush border membranes (BBM) of proximal kidney tubules. Previous studies have implicated meprins in Ischemic/Reperfusion (IR) induced acute kidney injury (AKI), and diabetic nephropathy (DN). However, the mechanisms by which meprins modulate kidney injury are not fully understood. Known meprin substrates include extracellular matrix (ECM) proteins (e.g. Collagen, Fibronectin, Laminin, and Nidogen‐1) and modulators of inflammation such as interleukin 1β (IL‐1β), IL‐6, pro‐IL‐18, monocyte chemoattractant protein‐1 (MCP‐1), and thymosin β4 – leading to release of the anti‐inflammatory peptide Ac‐SDKP. Furthermore, meprins are expressed in leukocytes (monocytes, macrophages), and play a role in leukocyte infiltration to sites of inflammation in the small intestines. Inflammation is an underlying cause of renal injury in both IR‐induced AKI and DN. The objective of the current study was to determine how meprin deficiency impacts extracellular matrix protein build up and leukocyte infiltration in unilateral ureteral obstruction (UUO)‐induced inflammation. Surgical procedures were used to ligate one ureter in twelve‐week‐old wild‐type (WT) and meprin β knockout male mice. The contralateral kidney served as the control for each mouse. At 7 days post‐UUO, the kidneys were perfused with saline to remove leukocytes present in kidney blood vessels and kidneys were harvested. Sections of the kidney tissue were snap frozen for protein extraction and some were paraffin embedded and sectioned for microscopy. For immunofluorescence F4/80, was used as a macrophage maker. The images were captured and the number of cells that stained positive for F4/80 counted. Western blot analysis was used to evaluate the levels of ECM proteins (fibronectin, laminin, and nidogen‐1) in the kidney lysates. Statistical analysis utilized 2‐way ANOVA (Graphpad Prism 7 software). Our data demonstrates increases in fibronectin protein levels in both WT and βKO kidneys subjected to UUO. However, UUO‐induced fibronectin increases were higher in βKO than in WT (p=0.0016 vs p=0.004). The baseline levels of nidogen‐1 were higher in WT mice (p=0.01) when compared to βKOs. At 7 days post‐UUO, the levels of nidogen‐1 increased significantly in the kidneys of βKO subjected to UUO (p=0.001), but not in WT kidneys. There were no significant differences in the baseline or post‐UUO levels of laminin in either genotype. Results from immunofluorescence showed that WT and meprin βKO kidneys subjected to UUO had significant increases in the number of macrophages in tubulo‐interstitial tissue when compared to control counterparts (≤0.007 and ≤0.001 respectively). Taken together, our data suggest that meprin expression/activity reduces the buildup of specific ECM proteins (fibronectin and nidogen‐1), thus reducing the fibrosis observed in inflammation. Furthermore, meprins modulate inflammation via enhanced leukocyte infiltration of (monocytes/macrophages).Support or Funding InformationNIH/National Institutes of General Medical Sciences (NIGMS) Grant #s SC3GM102049 and #SC1GM118271 to Elimelda Moige OngeriThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Long Noncoding RNA Zfas1 in Diabetic Cardiomyopathy

Fibrosis is a significant problem in diabetic cardiomyopathy (DCM). Both long noncoding RNAs (LncRNAs) and miRNAs (miR) play important roles in cellular processes. miR-9 targets important extracellular matrix(ECM) protein, such as fibronectin(FN) and collagen(Col), which are upregulated in DCM. The role of ZNFX1-AS1 (Zfas1), a LncRNA targeting miR-9 in DCM is not clear. We investigated Zfas1 in vitro and in vivo and its regulatory mechanism on ECM protein through miR-9. Microarrays were used to examine LncRNAs and miRNAs expression in ECs exposed to 25 mM glucose (HG) compare to 5 mM glucose and were validated by qPCR. Expression of ECM proteins targeted by miR-9 were examined. Luciferase assay was used to determine the interaction between miR-9 and its target genes. Cardiac tissues from streptozotocin-induced diabetic mice were examined similarly. The array and qPCR analysis showed glucose-induced upregulation of LncRNA Zfas1 and downregulation of miR-9 in ECs. FN mRNA, Col1&amp;4 mRNA and protein levels were increased in ECs treated with HG. Luciferase assay showed the binding of miR-9 to FN 3’-UTR and miR-9. Zfas1 silencing expression of its target genes expression in HG. Furthermore miR-9 mimics co-transfection with Zfas1 siRNA partially recovered these ECM protein expression. Interestingly, methylation blockade also reduced ECM protein expression. In the hearts of the diabetic animals, Zfas1 expression was increased and miR-9 was decreased and expression of transcripts regulated by miR-9 were increased. These studies indicate a novel glucose-induced mechanism of increased ECM protein production, which is regulated by Zfas1 through miR-9 in diabetes. Identifying such mechanisms may lead to potential RNA based treatment for diabetic complications. Disclosure B. Feng: None. S. Chen: None. S. Chakrabarti: None.

Poldip2 Affects Collagen I Accumulation by Regulating the Expression of Zyxin in Vascular Smooth Muscle Cells

Objectives: Polymerase delta interacting protein 2 (Poldip2) has previously been implicated in migration, proliferation and extracellular matrix (ECM) production in vascular smooth muscle cells. Thereby, regulation of Poldip2 protein expression is suggested to prevent the development and progression of diabetic macroangiopathy. We recently reported that Poldip2+/- mouse aortic smooth muscle cells (MASMs) exhibit higher β1-Integrin expression and activity of the PI3K/Akt/mTOR signaling pathway, leading to increased ECM protein synthesis. We further investigated the mechanism of Podlip2 in regulating the β1-Integrin expression. Approach and Results: Cell adhesion and cell signaling, below the focal adhesion, were investigated. Poldip2+/- MASMs (HET) showed both quicker cell attachment and detachment than Poldip2+/+ MASMs (WT). Paxillin, one of the components of focal adhesion, is expressed in all areas (from front to back) of lamellipodia in WT, on the other hand HET express the Paxillin only front area, which may induce weaker cell adhesion. The mature focal adhesion marker, Zyxin, expression is decreased in HET significantly. Intriguingly, the activity of RhoA is increased in HET and induced MRTF-A translocation from cytosol to nacreous. Conclusions: It is revealed that Poldip2 is positive regulator of Zyxin. HET has weak cell adhesion, and may induce Poldip2+/- MASMs exhibit higher β1-Integrin expression as a compensation against immature focal adhesion. Thus PI3K/Akt/mTOR signaling pathway is activated, leading to increased ECM protein synthesis. Translocation of MRTF-A from cytosol to the nucleus, may contribute to bind to transcriptional factor for Collagen I. This phenomenon is suggested as one more pathway, which Collagen I accumulates in HET. These findings have important implications for atherosclerotic vascular diseases such as diabetic macroangiopathy in which ECM accumulation plays a role. Disclosure M. Fujii: None. N. Sonoda: None. M. Okamoto: None. H. Morinaga: None. Y. Ogawa: Research Support; Self; AstraZeneca. Advisory Panel; Self; Gilead Sciences, Inc.. Research Support; Self; Japan Agency for Medical Research and Development, Japan Society for the Promotion of Science, Mitsubishi Tanabe Pharma Corporation, Mochida Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd.. K. Griendling: None.

Endoplasmic Reticulum Protein TXNDC5 Augments Myocardial Fibrosis by Facilitating Extracellular Matrix Protein Folding and Redox-Sensitive Cardiac Fibroblast Activation.

Cardiac fibrosis plays a critical role in the pathogenesis of heart failure. Excessive accumulation of extracellular matrix (ECM) resulting from cardiac fibrosis impairs cardiac contractile function and increases arrhythmogenicity. Current treatment options for cardiac fibrosis, however, are limited, and there is a clear need to identify novel mediators of cardiac fibrosis to facilitate the development of better therapeutics. Exploiting coexpression gene network analysis on RNA sequencing data from failing human heart, we identified TXNDC5 (thioredoxin domain containing 5), a cardiac fibroblast (CF)-enriched endoplasmic reticulum protein, as a potential novel mediator of cardiac fibrosis, and we completed experiments to test this hypothesis directly. The objective of this study was to determine the functional role of TXNDC5 in the pathogenesis of cardiac fibrosis. RNA sequencing and Western blot analyses revealed that TXNDC5 mRNA and protein were highly upregulated in failing human left ventricles and in hypertrophied/failing mouse left ventricle. In addition, cardiac TXNDC5 mRNA expression levels were positively correlated with those of transcripts encoding transforming growth factor β1 and ECM proteins in vivo. TXNDC5 mRNA and protein were increased in human CF (hCF) under transforming growth factor β1 stimulation in vitro. Knockdown of TXNDC5 attenuated transforming growth factor β1-induced hCF activation and ECM protein upregulation independent of SMAD3 (SMAD family member 3), whereas increasing expression of TXNDC5 triggered hCF activation and proliferation and increased ECM protein production. Further experiments showed that TXNDC5, a protein disulfide isomerase, facilitated ECM protein folding and that depletion of TXNDC5 led to ECM protein misfolding and degradation in CF. In addition, TXNDC5 promotes hCF activation and proliferation by enhancing c-Jun N-terminal kinase activity via increased reactive oxygen species, derived from NAD(P)H oxidase 4. Transforming growth factor β1-induced TXNDC5 upregulation in hCF was dependent on endoplasmic reticulum stress and activating transcription factor 6-mediated transcriptional control. Targeted disruption of Txndc5 in mice (Txndc5-/-) revealed protective effects against isoproterenol-induced cardiac hypertrophy, reduced fibrosis (by ≈70%), and markedly improved left ventricle function; post-isoproterenol left ventricular ejection fraction was 59.1±1.5 versus 40.1±2.5 (P<0.001) in Txndc5-/- versus wild-type mice, respectively. The endoplasmic reticulum protein TXNDC5 promotes cardiac fibrosis by facilitating ECM protein folding and CF activation via redox-sensitive c-Jun N-terminal kinase signaling. Loss of TXNDC5 protects against β agonist-induced cardiac fibrosis and contractile dysfunction. Targeting TXNDC5, therefore, could be a powerful new therapeutic approach to mitigate excessive cardiac fibrosis, thereby improving cardiac function and outcomes in patients with heart failure.

Notch1 regulates PTEN expression to exacerbate renal tubulointerstitial fibrosis in diabetic nephropathy by inhibiting autophagy via interactions with Hes1

Diabetic nephropathy (DN) is a serious clinical microvascular complication of diabetes mellitus. DN is characterized by the accumulation of extracellular matrix, resulting in progressive fibrosis leading to the loss of renal function. Notch1 and phosphatase and tensin homolog deleted on chromosome ten (PTEN) signaling have been associated with fibrosis. Autophagy serves as an essential regulator of tubular cellular homeostasis. However, how these molecules control the balance between fibrosis and autophagy, the main homeostatic mechanism regulating fibrosis, is not well understood. This association was confirmed using Notch1-siRNA in vitro, which prevented the increase in Hes1 and restored PTEN expression. In contrast, transfection with pHAGE-Hes1 repressed PTEN promoter-driven luciferase activity, implying a direct relationship between Hes1 and PTEN. The expression of Notch1 and Hes1 was increased in diabetic db/db mice by western blotting; in contrast, the expression of PTEN was decreased. Importantly, the dysregulation of these signaling molecules was associated with an increase in extracellular matrix proteins (Collagen-I and III) and the inhibition of autophagy. Similar results were evident in response to high glucose concentrations in vitro in the NRK-52e cells. Therefore, the high glucose concentrations present in diabetes promote fibrosis through the Notch1 pathway via Hes1, while inhibiting the PTEN and autophagy. In conclusion, the inhibition of PTEN by Notch1/Hes1 in response to high glucose concentration inhibits autophagy, which is associated with the progression of fibrosis. Therefore, these signaling molecules may represent novel therapeutic targets in diabetic nephropathy.

Proteomic Profiling of Human Uterine Fibroids Reveals Upregulation of the Extracellular Matrix Protein Periostin.

The central characteristic of uterine fibroids is excessive deposition of extracellular matrix (ECM), which contributes to fibroid growth and bulk-type symptoms. Despite this, very little is known about patterns of ECM protein expression in fibroids and whether these are influenced by the most common genetic anomalies, which relate to MED12. We performed extensive genetic and proteomic analyses of clinically annotated fibroids and adjacent normal myometrium to identify the composition and expression patterns of ECM proteins in MED12 mutation-positive and mutation-negative uterine fibroids. Genetic sequencing of tissue samples revealed MED12 alterations in 39 of 65 fibroids (60%) from 14 patients. Using isobaric tagged-based quantitative mass spectrometry on three selected patients (n = 9 fibroids), we observed a common set of upregulated (>1.5-fold) and downregulated (<0.66-fold) proteins in small, medium, and large fibroid samples of annotated MED12 status. These two sets of upregulated and downregulated proteins were the same in all patients, regardless of variations in fibroid size and MED12 status. We then focused on one of the significant upregulated ECM proteins and confirmed the differential expression of periostin using western blotting and immunohistochemical analysis. Our study defined the proteome of uterine fibroids and identified that increased ECM protein expression, in particular periostin, is a hallmark of uterine fibroids regardless of MED12 mutation status. This study sets the foundation for further investigations to analyze the mechanisms regulating ECM overexpression and the functional role of upregulated ECM proteins in leiomyogenesis.