Mitogenic Growth Factors Research Articles

Essential growth factor receptors for fibroblast homeostasis and activation: Fibroblast Growth Factor Receptor (FGFR), Platelet Derived Growth Factor Receptor (PDGFR), and Transforming Growth Factor β Receptor (TGFβR).

Fibroblasts are cells of mesenchymal origin that are found throughout the body. While these cells have several functions, their integral roles include maintaining tissue architecture through the production of key extracellular matrix components, and participation in wound healing after injury. Fibroblasts are also key mediators in disease progression during fibrosis, cancer, and other inflammatory diseases. Under these perturbed states, fibroblasts can activate into inflammatory fibroblasts or contractile myofibroblasts. Fibroblasts require various growth factors and mitogenic molecules for survival, proliferation, and differentiation. While the activity of mitogenic growth factors on fibroblasts in vitro was characterized as early as the 1970s, the proliferation and differentiation effects of growth factors on these cells in vivo are unclear. Recent work exploring the heterogeneity of fibroblasts raises questions as to whether all fibroblast cell states exhibit the same growth factor requirements. Here, we will examine and review existing studies on the influence of fibroblast growth factor receptors (FGFRs), platelet-derived growth factor receptors (PDGFRs), and transforming growth factor β receptor (TGFβR) on fibroblast cell states.

Essential growth factor receptors for fibroblast homeostasis and activation

Fibroblasts are cells of mesenchymal origin that are found throughout the body. While these cells have several functions, their integral roles include maintaining tissue architecture through the production of key extracellular matrix components, and participation in wound healing after injury. Fibroblasts are also key mediators in disease progression during fibrosis, cancer, and other inflammatory diseases. Under these perturbed states, fibroblasts can activate into inflammatory fibroblasts or contractile myofibroblasts. Fibroblasts require various growth factors and mitogenic molecules for survival, proliferation, and differentiation. While the activity of mitogenic growth factors on fibroblasts in vitro was characterized as early as the 1970s, the proliferation and differentiation effects of growth factors on these cells in vivo are unclear. Moreover, recent work exploring the heterogeneity of fibroblasts raises questions as to whether all fibroblast cell states exhibit the same growth factor requirements. Here, we will examine and review existing growth factors known to influence fibroblast homeostasis to begin unpacking the potential growth factors that may influence in vivo fibroblast cell states.

Overexpression and Purification of Mitogenic and Metabolic Fibroblast Growth Factors.

Fibroblast growth factors (FGFs) are proteins with a vast array of biological activity, such as cell development and repair, glucose and bile acid metabolisms, and wound healing. Due to their critical and diverse physiological functions, FGFs are believed to possess potential as therapeutic agents for many diseases and conditions that warrant further investigations. Thus, a simple, cost-efficient method to purify these biologically active signaling proteins is desirable. Herein, we introduce such techniques to purify FGFs that possess either high heparin-binding affinity or low to no heparin-binding affinity. This method takes advantage of the high affinity toward heparin sulfate from paracrine FGF1 to isolate the targeted protein. It also accounts for FGF members that have low heparin affinity, such as the metabolic FGFs, by introducing poly-histidine tags in the recombinant protein in combination with the immobilized metal affinity chromatography. Subsequently, the purified FGF products are separated from the other small protein by high-speed centrifugation. Products are then subjected to other biophysical experiments like SDS-PAGE, mass spectrometry, circular dichroism, intrinsic fluorescence, isothermal titration calorimetry, differential scanning calorimetry, and biological cell activity assay to confirm that the target proteins are purified with intact native conformation and no significant change in the intrinsic characteristics and biological activities.

MiRNAs from Inflamed Gingiva Link Gene Signaling to Increased MET Expression

Several array-based microRNA (miRNA) expression studies independently showed increased expression of miRNAs hsa-miR-130a-3p, -142-3p, -144-3p, -144-5p, -223-3p, -17-5p, and -30e-5p in gingiva affected by periodontal inflammation. We aimed to determine direct target genes and signaling pathways regulated by these miRNAs to identify processes relevant to gingival inflammatory responses and tissue homeostasis. We transfected miRNA mimics (mirVana) for each of the 7 miRNAs separately into human primary gingival fibroblasts cultured from 3 different donors. Following RNA sequencing, differential gene expression and second-generation gene set enrichment analyses were performed. miRNA inhibition and upregulation was validated at the transcript and protein levels using quantitative reverse transcriptase polymerase chain reaction, Western blotting, and reporter gene assays. All 7 miRNAs significantly increased expression of the gene MET proto-oncogene, receptor tyrosine kinase (MET). Expression of known periodontitis risk genes CPEB1, ABCA1, and ATP6V1C1 was significantly repressed by hsa-miR-130a-3p, -144-3p, and -144-5p, respectively. The genes WASL, ENPP5, ARL6IP1, and IDH1 showed the most significant and strongest downregulation after hsa-miR-142-3p, -17-5p, -223-3p, and -30e-5p transfection, respectively. The most significantly regulated gene set of each miRNA related to cell cycle (hsa-miRNA-144-3p and -5p [Padj = 4 × 10−40 and Padj = 4 × 10−6], -miR-17-5p [Padj = 9.5 × 10−23], -miR-30e-5p [Padj = 8.2 × 10−18], -miR-130a-3p [Padj = 5 × 10−15]), integrin cell surface interaction (-miR-223-3p [Padj = 2.4 × 10−7]), and interferon signaling (-miR-142-3p [Padj = 5 × 10−11]). At the end of acute inflammation, gingival miRNAs bring together complex regulatory networks that lead to increased expression of the gene MET. This underscores the importance of mesenchymal cell migration and invasion during gingival tissue remodeling and proliferation in restoring periodontal tissue homeostasis after active inflammation. MET, a receptor of the mitogenic hepatocyte growth factor fibroblast secreted, is a core gene of this process.

Free-standing multilayer films as growth factor reservoirs for future wound dressing applications

Chronic skin wounds place a high burden on patients and health care systems. The use of angiogenic and mitogenic growth factors can facilitate the healing but growth factors are quickly inactivated by the wound environment if added exogenously. Here, free-standing multilayer films (FSF) are fabricated from chitosan and alginate as opposing polyelectrolytes in an alternating manner using layer-by-layer technique. One hundred bilayers form an about 450 μm thick, detachable free-standing film that is subsequently crosslinked by either ethyl (dimethylaminopropyl) carbodiimide combined with N-hydroxysuccinimide (E-FSF) or genipin (G-FSF). The characterization of swelling, oxygen permeability and crosslinking density shows reduced swelling and oxygen permeability for both crosslinked films compared to non-crosslinked films (N-FSF). Loading of fibroblast growth factor 2 (FGF2) into the films results in a sustained release from crosslinked FSF in comparison to non-crosslinked FSF. Biocompatibility studies in vitro with human dermal fibroblasts cultured underneath the films demonstrate increased cell growth and cell migration for all films with and without FGF2. Especially G-FSF loaded with FGF2 greatly increases cell proliferation and migration. In vivo biocompatibility studies by subcutaneous implantation in mice show that E-FSF causes an inflammatory tissue response that is absent in the case of G-FSF. N-FSF also represents a biocompatible film but shows early degradation. All FSF possess antibacterial properties against gram+ and gram- bacteria demonstrated by an agar diffusion disc assay. In summary, FSF made of alginate and chitosan crosslinked with genipin can act as a reservoir for the sustained release of FGF2, possessing high biocompatibility in vitro and in vivo. Moreover, G-FSF promotes growth and migration of human dermal fibroblasts and has antibacterial properties, which makes it an interesting candidate for bioactive wound.

Recombinant production of growth factors for application in cell culture

SummaryCulturing eukaryotic cells has widespread applications in research and industry, including the emerging field of cell-cultured meat production colloquially referred to as “cellular agriculture”. These applications are often restricted by the high cost of growth medium necessary for cell growth. Mitogenic protein growth factors (GFs) are essential components of growth medium and account for upwards of 90% of the total costs. Here, we present a set of expression constructs and a simplified protocol for recombinant production of functionally active GFs, including FGF2, IGF1, PDGF-BB, and TGF-β1 in Escherichia coli. Using this E. coli expression system, we produced soluble GF orthologs from species including bovine, chicken, and salmon. Bioactivity analysis revealed orthologs with improved performance compared to commercially available alternatives. We estimated that the production cost of GFs using our methodology will significantly reduce the cost of cell culture medium, facilitating low-cost protocols tailored for cultured meat production and tissue engineering.

Abstract 1674: FGF8 as a modulator of Wilms' tumor in vitro growth and longevity

Abstract Wilms’ tumor is the most common pediatric renal tumor accounting for 6-7% of all childhood cancers. Development of faithful Wilms’ tumor cell lines is needed to identify novel therapeutics. Currently, established Wilms’ tumor cell lines that are suitable for high throughput studies only represent anaplastic Wilms, a rare subtype. In contrast, cell lines representing the more common favorable histology Wilms’ tumor (FHWT) often have poor proliferative capacity and are unable to passage long-term (e.g. >20 passages). For this reason, there is a need to establish robust cell lines representing FHWT. We hypothesize that there are core factors missing from the cell culture medium which prevent proper growth signaling and proliferation. We first performed bulk RNA-sequencing from patients and associated cell lines at Aflac Cancer and Blood Disorders Center with an underlying diagnosis of FHWT. Transcriptomic analyses showed that the reduced growth and proliferation in these FHWT cell lines may be due to reduced presence of the mitogenic fibroblast growth factor 8 (FGF8) in the cell culture environment as compared to the tumor environment. Wilms’ tumor has notably high FGF8 expression levels compared to all other pediatric tumors, implicating FGF8 as a potential driving factor in tumor maintenance. To characterize the phenotypic effects of FGF8 reintroduction in short term FHWT cell lines, we both endogenously overexpressed FGF8 via a lentiviral vector and ectopically added recombinant FGF8 to the cell culture medium. Qualitatively, endogenous overexpression of FGF8 induced distinct morphological changes in cells, converting the enlarged and flattened cells into more focal cells resembling proliferative early passage cells. Quantitatively, overexpression of FGF8 in one favorable histology cell line led to an increased rate of cellular proliferation and extended the cellular longevity by seven passages (47% above baseline). RNA-sequencing analysis of these FGF8 overexpressing cells implicated the anti-apoptotic gene, NTSR1, and an extracellular matrix protein, SNED1, as potential downstream targets of FGF8 overexpression. However, ectopic addition of recombinant FGF8 protein to the cellular medium had less pronounced effects, with seemingly cell-line specific effects. These data demonstrate FGF8 as possibly being required for survival in certain Wilms’ tumor cell lines that can be modulated to influence in vitro proliferation and longevity. Citation Format: Garrett Cooper, Benjamin Lee, Nate Smyth, Karuna Mittal, Taylor Lawrence, Hunter Jonus, Jenny Shim, Kelly Goldsmith, Andrew Hong. FGF8 as a modulator of Wilms' tumor in vitro growth and longevity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1674.

Reproductive health and stress fracture risk: a wearables solution

Introduction: Insulin-like growth factor 1 (IGF-I) is a mitogenic and metabolic growth factor that can act both systemically and locally, being produced predominantly in the liver but also by other tissues including the brain, kidneys, muscle, and bone1,2. IGF-I is well-established as an important mediator of bone formation, remodelling, and repair and can be easily measured in serum. IGF-I stimulates osteoblast proliferation, bone matrix protein synthesis, alkaline phosphatase activity and differentiation. Loss of IGF-I results in decreased long bone size, reduced growth plates, and delayed formation of secondary ossification centers in mice. Conversely, transgenic mice with osteoblast-targeted overexpression of IGF-I demonstrate an accelerated bone formation rate with increased bone mineral density (BMD) and trabecular and cortical bone volume. The essential nature of IGF-I in bone development is recapitulated in humans. Individuals with disruptions in the IGF-I gene causing reduced serum IGF-I concentrations have a severe reduction in peak BMD compared to normal age-matched individuals. Methods: This presentation will provide an overview of the relationship between the IGF-I system and bone health and remodelling, with a particular emphasis toward military relevance and utility of measuring IGF-I during military training2,3. Because the IGF-I system is involved in bone formation, remodelling, and repair, female recruits sustaining stress fractures during military training would be of special interest with regard to differential circulating concentrations of IGF-I system components compared to their uninjured female counterparts. Moreover, serum measurements of the IGF-I system could possibly provide further insight into stress fracture injury risk. Conclusions: Preliminary data3 suggest that circulating IGF-I system components are differentially expressed in females sustaining stress fractures during military BT. Additionally, data suggest that the bioavailable IGF-I response to strenuous exercise may differ in female recruits susceptible to stress fracture injury when compared to non-injured recruits. This suggests the possibility that increased susceptibility to stress fracture injury may be attributed to differences in the IGF-I system response to physical training (i.e., increased susceptibility to stress fracture may be due to a blunted bioavailable IGF-I response to training or increased basal levels of certain IGFBPs). However, the underlying mechanism for the differences in IGF-I system component concentrations between recruits sustaining stress fractures and uninjured counterparts is still unclear and warrants further investigation. References 1Nindl BC, Pierce JR. Insulin-like growth factors I as a biomarker of health, fitness, and training status. Med Sci Sports Exerc 2010; 42(1):39-49. https://doi.org/10.1249/mss.0b013e3181b07c4d 2Nindl BC, McClung JP, Miller JK, et al. Bioavailable IGF-I is associated with fat-free mass gains after physical training in women. Med Sci Sports Exerc 2011; 43(5):793-799. https://doi.org/10.1249/MSS.0b013e31820065.ea 3Strohbach CA, Scofield DE, Nindl BC, et al. Female recruits sustaining stress fractures during military basic training demonstrate differential concentrations of circulating IGF-I system components: a preliminary study. Growth Horm IGF Res 2012; 22(5):151-157. https://doi.org/10.1016/j.ghir.2012.04.007

CLINICAL PECULIARITIES AND PATHOGENETIC DETERMINANTS OF DEVELOPMENT AND PROGRESSION OF CHRONIC PULMONARY HEART WITH ARTERIAL HYPERTENSION

The aim: To investigate the clinical and pathogenetic peculiarities of formation and course of CCP and the relationship between clinical, hemodynamic and neurohumoral factors of comorbidity development in COPD combined with arterial hypertension (AH). Materials and methods: The object of the study were 484 patients with COPD. Among them, 350 patients with CCP as a result of cardiac insufficiency/severe congestive heart failure (COPD III-IV) out of aggravation, combined with AH II stage (non-symptomatic organ damage) and 1 - 3 grades, including 55 patients (43 men, 12 women) with compensated CCP (average age 43.7 ± 3.4 years), and 295 patients (212 men and 83 women) with decompensated CCP and chronic heart failure (CHF), average age 63.2 ± 8.9 years. Results: It was found that the development and progression of the left and right CHF in patients with CCP combined with AH occurs due to the disorders of the central hemodynamic, progression of pulmonary hypertension, bronchial obstruction syndrome, neurohumoral and systemic immunoinflammatory activation, disorders of endothelial regulation of vascular tension, overproduction of epithelial and mitogenic growth factors, inducers of apoptosis, and is accompanied by increasing levels of natriuretic peptides. Conclusions: The main pathogenetic formation mechanisms of the heart failure on the background of CCP combined with AH are: neurohumoral and systematic immune-inflammatory activation with the development of endothelial dysfunction and (neo)angiogenesis, induction of pathological apoptosis, increase in the intrathoracic pressure, and deposition of blood in the extrathoracic tissues, which result in pulmonary and systemic hypertension, metabolic and hemodynamic remodelling and heart dysfunction.

Autophagy-Related Activation of Hepatic Stellate Cells Reduces Cellular miR-29a by Promoting Its Vesicular Secretion.

Background & AimsLiver fibrosis arises from long-term chronic liver injury, accompanied by an accelerated wound healing response with interstitial accumulation of extracellular matrix (ECM). Activated hepatic stellate cells (HSC) are the main source for ECM production. MicroRNA29a (miR-29a) is a crucial antifibrotic miRNA that is repressed during fibrosis, resulting in up-regulation of collagen synthesis.MethodsIntracellular and extracellular miRNA levels of primary and immortalized myofibroblastic HSC in response to profibrogenic stimulation by transforming growth factor β (TGFβ) or platelet-derived growth factor-BB (PDGF-BB) or upon inhibition of vesicular transport and autophagy processes were determined by quantitative polymerase chain reaction. Autophagy flux was studied by electron microscopy, flow cytometry, immunoblotting, and immunocytochemistry. Hepatic and serum miR-29a levels were quantified by using both liver tissue and serum samples from a cohort of chronic hepatitis C virus patients and a murine CCl4 induced liver fibrosis model.ResultsIn our study, we show that TGFβ and PDGF-BB resulted in decrease of intracellular miR-29a and a pronounced increase of vesicular miR-29a release into the supernatant. Strikingly, miR-29a vesicular release was accompanied by enhanced autophagic activity and up-regulation of the autophagy marker protein LC3. Moreover, autophagy inhibition strongly prevented miR-29a secretion and repressed its targets’ expression such as Col1A1. Consistently, hepatic miR-29a loss and increased LC3 expression in myofibroblastic HSC were associated with increased serum miR-29a levels in CCl4-treated murine liver fibrosis and specimens of hepatitis C virus patients with chronic liver disease.ConclusionsWe provide evidence that activation-associated autophagy in HSC induces release of miR-29a, whereas inhibition of autophagy represses fibrogenic gene expression in part through attenuated miR-29a secretion.

Impact of Human Epidermal Growth Factor on Tissue-Engineered Skeletal Muscle Structure and Function.

Skeletal muscle tissue engineering technologies have the potential to treat volumetric muscle loss (VML) by growing exogenous muscle tissue. However, there has been limited success in engineering human cell-sourced skeletal muscle with structure and function comparable to native adult human muscle. The use of growth factors at optimal concentrations and delivery times is critical in enhancing the in vitro myogenesis of satellite cells used in engineered skeletal muscle. The mitogenic protein human epidermal growth factor (hEGF) is of particular interest because it enhances satellite cell proliferation and sarcomeric structure formation in myogenic cell cultures. In this study, we used our scaffold-free tissue-engineered skeletal muscle units (SMUs) to examine the effects of hEGF on the structure and function of human cell-sourced engineered skeletal muscle. During our established SMU fabrication process, human muscle cell isolates were exposed to media treated with 7.5 nM hEGF at three different time spans during the 21-day cell culture period: 0 to 6 days postseeding (hEGF-treated Muscle Growth Media [MGM] Only), 7 to 21 days postseeding (hEGF-treated Muscle Differentiation Media (MDM) Only), and 0 to 21 days postseeding (hEGF-treated MGM+MDM). Control cell cultures were fed standard MGM and MDM (no hEGF treatment). During the fabrication process, light microscopy was used to examine proliferation and differentiation of myogenic cells in the monolayer. After SMU formation, the three-dimensional constructs underwent tetanic force production measurements to evaluate contractile function and immunohistochemical staining to examine SMU structure. Results indicated that hEGF administration impacted myogenesis, by increasing myotube diameter in hEGF-treated MGM only and hEGF-treated MDM-only cell cultures, and by increasing myotube density in hEGF-treated MGM+MDM cultures. The exposure of myogenic cells to hEGF during any time period of the fabrication process led to a significant increase in SMU myosin heavy-chain content. SMUs exposed to hEGF-treated MDM and hEGF-treated MGM+MDM exhibited greater cross-sectional areas and more organized sarcomeric structure. Furthermore, hEGF-treated MGM+MDM SMUs displayed significantly enhanced contractile function compared with controls, indicating advanced functional maturation. In conclusion, hEGF supplementation in human primary myogenic cell cultures advances tissue-engineered skeletal muscle structural and functional characteristics. Impact statement Our research suggests that human epidermal growth factor (hEGF) serves as a critical growth factor in enhancing in vitro skeletal muscle cell proliferation and differentiation during myogenesis and advances human skeletal muscle engineered tissues toward a more native adult skeletal muscle phenotype. Understanding the impact of hEGF on engineered skeletal muscle function and structure is valuable in determining the optimal culture conditions for the development of tissue engineering-based therapies for volumetric muscle loss.

Role of Insulin-Like Growth Factor Receptor 2 across Muscle Homeostasis: Implications for Treating Muscular Dystrophy.

The insulin-like growth factor 2 receptor (IGF2R) plays a major role in binding and regulating the circulating and tissue levels of the mitogenic peptide insulin-like growth factor 2 (IGF2). IGF2/IGF2R interaction influences cell growth, survival, and migration in normal tissue development, and the deregulation of IGF2R expression has been associated with growth-related disease and cancer. IGF2R overexpression has been implicated in heart and muscle disease progression. Recent research findings suggest novel approaches to target IGF2R action. This review highlights recent advances in the understanding of the IGF2R structure and pathways related to muscle homeostasis.

Research advances in repair of growth plate injury

Growth plate, the developmental center of endochondral osteogenesis, can be divided morphologically and functionally into a resting zone, a proliferative zone, a prehypertrophic zone and a hypertrophic zone. Injuries to growth plate often lead to bone growth defects including limb length discrepancy and angulation deformity in children. Currently, their orthopedic corrective surgeries are invasive and limitedly effective and no effective biotherapy has been available. Previous studies on animal models of growth plate damage have investigated the related cellular and molecular events in the repair of damaged growth plates in the 4 distinct inflammatory, fibrogenic, osteogenic and remodeling phases. Related molecules involved in the regulation of the above processes, such as inflammatory cytokines tumor necrosis factor alpha, mitogenic platelet-derived growth factor and bone morphogenetic protein, are found to participate in the regulation of growth plate injury. Exploration of the mechanisms may provide new targets for biotherapy. In addition, development of cartilage tissue engineering, especially application of mesenchymal stem cells, also provides potential interventions for growth plate injury. Key words: Tissue engineering; Wounds and injuries; Bone marrow cells; Growth plate; Chondrogenesis

Placental endocrine malfunction programs ovarian defects in female murine offspring

Development of the placenta must always be in advance of that of the embryo. Evidence from domestic species demonstrates that the placenta is capable of stimulating its own development through a signalling dialogue with the endometrial glands. Placental lactogens produced by the trophoblast lead to increased expression and release of uterine secretions and mitogenic growth factors, including epidermal growth factor, that have a close temporal and spatial relationship with trophoblast proliferation. Here, we review evidence that an equivalent mechanism operates in the human. The same repertoire of receptors is present on the endometrial gland cells, and the epithelial cells have long been known to adopt a hypersecretory phenotype following an implantation. Furthermore, early pregnancy hormones stimulate the secretion of glycodelin-A and osteopontin, two ‘uterine milk proteins’ that have multiple potential effects at the maternal-placental interface, from organoid cultures derived from endometrial glands. Prolactin appears to be an important stimulant, but unlike in domestic species the human trophoblast does not secrete this hormone. Instead, it is a major product of decidual cells. Hence, complications of pregnancy that have their pathophysiological roots in deficient trophoblast proliferation may be due primarily to problems of decidualisation. Ensuring the endometrium is in an optimal state pre-conceptionally should therefore be a priority for women's health. Trophoblast stemness and proliferative capacity show a sharp decline at the switch from histotrophic to haemotrophic nutrition. This may reflect the increase in oxygen concentration or loss of growth factor support. Either way, there are implications for adaptive growth of the organ.

Abstract 4308: Phosphatidic acid binds to ERK3 and stimulates phosphorylation of the ERK3 activation loop

Abstract Protein kinases modulate the activity of other proteins through phosphorylation and themselves are often regulated by phosphorylation of their activation loop. Extracellular signal-Regulated Kinase 3 (ERK3) is an atypical member of the ERK family of Mitogen-Activated Protein Kinases (MAPK), with a SerGlu-Gly (SEG) motif in its activation loop rather than the canonical Thr-Xaa-Tyr (TXY) motif of ERK1/2. Activation loop phosphorylation of ERK3 is not regulated by the signals known to activate ERK1/2, such as mitogenic growth factors. The cellular stimuli activating ERK3 kinase are virtually unknown. Some kinases, including mTOR and p21 activated kinase 1 and 2 (PAK1/2), are stimulated by binding to certain phospho-lipid signaling molecules, such as phosphatidic acids. Here, we show ERK3 binds to specific phospholipids in vitro, and the C-terminal domain may be important for this interaction. In addition, ERK3 can localize to the cell membrane. Further, phosphatidic acid stimulated phosphorylation of the activation loop in ERK3. Together, these results suggest that ERK3 kinase activity might be upregulated by specific phosphatidic acid species in cells. Citation Format: Amanda Myers, Hitham Aldharee, Shimpi Bedi, Weiwen Long. Phosphatidic acid binds to ERK3 and stimulates phosphorylation of the ERK3 activation loop [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4308.

P53‐Mediated Activities in NS‐5 Neural Stem Cells: Effects of Ethanol

Transforming growth factor (TGF) β1 and ethanol (EtOH) powerfully inhibit the proliferation, DNA repair, and survival of neural stem cells (NSCs). The present study tests the hypothesis that the EtOH-induced DNA damage response is mediated through p53 pathways and influenced by growth factor signals. Cultures of nonimmortalized NSCs, NS-5 cells, were transfected with p53 siRNA, exposed to either the mitogenic fibroblast growth factor (FGF) 2 or antimitogenic TGFβ1, and to EtOH. Stage-specific cellular and genomic responses were examined. p53 status, EtOH exposure, and growth factor significantly affected the expression of transcripts related to the DNA damage response (including those coding for excision repair proteins), mitotic promoters, and regulators of cell death via the tumor necrosis factor pathway. There were significant compensatory increases in p53 family members, p63 and p73, notably in regard to the regulation of cell cycle restriction and apoptosis. Treatment with p53 siRNA potentiated EtOH- and TGFβ1-induced changes in the numbers of proliferating NSCs and increased the proportion of NSCs expressing the apoptotic marker annexin V. Thus, it appears that EtOH and TGFβ1 affect proliferation, DNA repair, and survival of NSCs via p53-mediated activities.

161. Placental development, blood flow and pre-eclampsia; A role for the endometrium?

Placental stress, induced by maternal malperfusion, is believed to be at the epicentre of the pathophysiology of pre-eclampsia, at least for the early-onset sub-type. Deficient remodelling of the spiral arteries has been well documented in the placental bed, with the degree correlating with time of onset of the syndrome. Despite this progress, the cause for the deficiency is still not known. Impaired invasion by the extravillous trophoblast cells essential to the remodelling process has been implicated, as have aberrant interactions between these cells and those of the maternal immune system present in the decidua. Another possibility is that early development of the placenta post-implantation is abnormal, resulting in an impoverished supply of extravillous trophoblast cells. Although this hypothesis has received little attention to date, recent transcriptional analyses have revealed abnormalities in decidualisation in women prior to, and after, pre-eclampsia. Initial growth of the placenta is stimulated by histotroph from the endometrial glands, which contains numerous mitogenic growth factors as well as glucose, lipids and other nutrients. The secretory activity of the glands increases in early pregnancy in response to lactogenic hormones produced by the placenta. However, details of the signalling pathways involved are unknown due to the inaccessibility of the tissues concerned. The recent derivation of endometrial organoids now allows these pathways to be dissected, and prolactin is emerging as a strong stimulant of glandular secretion. Prolactin is secreted by decidual cells, and so may link the transcriptomic data with the supply of extravillous trophoblast cells and deficient arterial remodelling. If the hypothesis were proved correct, the translational impact would be to emphasise the importance of ensuring optimal endometrial function prior to conception. This would have the great advantage of preventing the pre-eclampsia and other placental-related complications at their outset, rather than trying to treat these disorders once established.

A Cell Culture Approach to Optimized Human Corneal Endothelial Cell Function.

PurposeCell-based therapies to replace corneal endothelium depend on culture methods to optimize human corneal endothelial cell (HCEC) function and minimize endothelial-mesenchymal transition (EnMT). Here we explore contribution of low-mitogenic media on stabilization of phenotypes in vitro that mimic those of HCECs in vivo.MethodsHCECs were isolated from cadaveric donor corneas and expanded in vitro, comparing continuous presence of exogenous growth factors (“proliferative media”) to media without those factors (“stabilizing media”). Identity based on canonical morphology and expression of surface marker CD56, and function based on formation of tight junction barriers measured by trans-endothelial electrical resistance assays (TEER) were assessed.ResultsPrimary HCECs cultured in proliferative media underwent EnMT after three to four passages, becoming increasingly fibroblastic. Stabilizing the cells before each passage by switching them to a media low in mitogenic growth factors and serum preserved canonical morphology and yielded a higher number of cells. HCECs cultured in stabilizing media increased both expression of the identity marker CD56 and also tight junction monolayer integrity compared to cells cultured without stabilization.ConclusionsHCECs isolated from donor corneas and expanded in vitro with a low-mitogenic media stabilizing step before each passage demonstrate more canonical structural and functional features and defer EnMT, increasing the number of passages and total canonical cell yield. This approach may facilitate development of HCEC-based cell therapies.

Palmitic acid dysregulates the Hippo–YAP pathway and inhibits angiogenesis by inducing mitochondrial damage and activating the cytosolic DNA sensor cGAS–STING–IRF3 signaling mechanism

Impaired angiogenesis and wound healing carry significant morbidity and mortality in diabetic patients. Metabolic stress from hyperglycemia and elevated free fatty acids have been shown to inhibit endothelial angiogenesis. However, the underlying mechanisms remain poorly understood. In this study, we show that dysregulation of the Hippo-Yes-associated protein (YAP) pathway, an important signaling mechanism in regulating tissue repair and regeneration, underlies palmitic acid (PA)-induced inhibition of endothelial angiogenesis. PA inhibited endothelial cell proliferation, migration, and tube formation, which were associated with increased expression of mammalian Ste20-like kinases 1 (MST1), YAP phosphorylation/inactivation, and nuclear exclusion. Overexpression of YAP or knockdown of MST1 prevented PA-induced inhibition of angiogenesis. When searching upstream signaling mechanisms, we found that PA dysregulated the Hippo-YAP pathway by inducing mitochondrial damage. PA treatment induced mitochondrial DNA (mtDNA) release to cytosol, and activated cytosolic DNA sensor cGAS-STING-IRF3 signaling. Activated IRF3 bound to the MST1 gene promoter and induced MST1 expression, leading to MST1 up-regulation, YAP inactivation, and angiogenesis inhibition. Thus, mitochondrial damage and cytosolic DNA sensor cGAS-STING-IRF3 signaling are critically involved in PA-induced Hippo-YAP dysregulation and angiogenesis suppression. This mechanism may have implication in impairment of angiogenesis and wound healing in diabetes.

Feedback regulation of steady-state epithelial turnover and organ size.

Epithelial organs undergo steady-state turnover throughout adult life, with old cells being continually replaced by the progeny of stem cell divisions. To avoid hyperplasia or atrophy, organ turnover demands strict equilibration of cell production and loss. However, the mechanistic basis of this equilibrium is unknown. Here we show that robustly precise turnover of the adult Drosophila intestine arises through a coupling mechanism in which enterocyte apoptosis breaks feedback inhibition of stem cell division. Healthy enterocytes inhibit stem cell division through E-cadherin, which prevents secretion of mitogenic epidermal growth factors (EGFs) by repressing transcription of the EGF maturation factor rhomboid. Individual apoptotic enterocytes promote divisions by loss of E-cadherin, which releases cadherin-associated β-catenin (Armadillo in Drosophila) and p120-catenin to induce rhomboid. Induction of rhomboid in the dying enterocyte triggers activation of the EGF receptor (Egfr) in stem cells within a discrete radius. When we blocked apoptosis, E-cadherin-controlled feedback suppressed divisions, and the organ retained the same number of cells. When we disrupted feedback, apoptosis and divisions were uncoupled, and the organ developed either hyperplasia or atrophy. Together, our results show that robust cellular balance hinges on the obligate coupling of divisions to apoptosis, which limits the proliferative potential of a stem cell to the precise time and place at which a replacement cell is needed. In this way, localized cell-cell communication gives rise to tissue-level homeostatic equilibrium and constant organ size.