Endothelial dysfunction and/or injury is pivotal to the development of cardiovascular and inflammatory pathology. Endothelial dysfunction and/or injury has been described in Human Immunodeficiency Virus (HIV) infection. Elaboration of circulating markers of endothelial activation, such as soluble adhesion molecules and procoagulant proteins, occurs in HIV infection. Certain endothelial cells, such as those lining liver sinusoids, human umbilical vein endothelial cells, bone marrow stromal endothelial cells or brain microvascular endothelial cells, have been shown to be variably permissive for HIV infection. Entry of virus into endothelial cells may occur via CD4 antigen or galactosyl-ceramide receptors. Other mechanisms of entry including chemokine receptors have been proposed. Nevertheless, endothelial activation may also occur in HIV infection either by cytokines secreted in response to mononuclear or adventitial cell activation by virus or else by the effects of the secreted HIV-associated proteins, gp 120 (envelope glycoprotein) and Tat (transactivator of viral replication) on endothelium. Enhanced adhesiveness of endothelial cells, endothelial cell proliferation and apoptosis as well as activation of cytokine secretion have all been demonstrated. Synergy between select inflammatory cytokines and viral proteins in inducing endothelial injury has been shown. In HIV infection, dysfunctional or injured endothelial cells potentiate tissue injury, inflammation and remodeling, and accelerate the development of cardiovascular disease.

Indiana University-Purdue University Indianapolis (IUPUI) / Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis is a central readout of endothelial progenitor cell functionality. However, current assays lack kinetic measurements. To address this issue, new approaches were developed to quantitatively assess in vitro endothelial colony forming cell (ECFC) network formation in real time. Eight parameters of network structure were quantified using novel Kinetic Analysis of Vasculogenesis (KAV) software. KAV assessment of structure complexity identified two phases of network formation. This observation guided the development of additional vasculogenic readouts, including a tissue cytometry approach to quantify the frequency and localization of dividing ECFCs within cell networks. Additionally, FIJI TrackMate was used to quantify ECFC displacement and speed at the single cell level during network formation. These novel approaches were then applied to determine how intrauterine exposure to maternal type 2 diabetes mellitus (T2DM) impairs fetal ECFC vasculogenesis, and whether increased Transgelin 1 (TAGLN) expression in ECFCs from pregnancies complicated by gestational diabetes (GDM) was sufficient to impair vasculogenesis. Fetal ECFCs exposed to maternal T2DM formed fewer initial network structures, which were not stable over time. Correlation analyses identified that ECFC samples with greater division in branches formed fewer closed network structures and that reductions in ECFC movement decreased structural connectivity. To identify specific cellular mechanisms and signaling pathways altered in ECFCs following intrauterine GDM exposure, these new techniques were also applied in TAGLN expression studies. Similarly, ECFCs from GDM pregnancies and ECFCs overexpressing TAGLN exhibited impaired vasculogenesis and decreased migration. Both ECFCs from GDM pregnancies as well as ECFCs over expressing TAGLN exhibited increased phosphorylation of myosin light chain. Reduction of myosin light chain phosphorylation via Rho kinase inhibition increased ECFC migration; therefore, increased TAGLN was sufficient to impair ECFC vasculogenic function. Overall, identification of these novel phenotypes provides evidence for the molecular mechanisms contributing to aberrant ECFC vasculogenesis. Determining how intrauterine exposure to maternal T2DM and GDM alters fetal ECFC function will enable greater understanding of the chronic vascular pathologies observed in children from pregnancies complicated by diabetes mellitus.

Indiana University-Purdue University Indianapolis (IUPUI) / Depressed adults are at increased risk of developing atherosclerotic cardiovascular disease (CVD). However, heterogeneity in the depressed population engenders a key question: Are there subgroups of depressed adults at greater risk of developing CVD? Because other affective traits – i.e., anxiety, hostility/anger, and low trait positive affect – have also been associated with increased CVD risk, depressed adults with higher levels of these co-occurring affective traits may have an elevated risk of developing CVD. Consequently, the present study’s first aim was to examine, in depressed adults, which affective traits (depression, anxiety, hostility/anger, or low positive affect) are associated with endothelial function, a marker of cumulative CVD risk. In addition, because the other affective traits overlap with depressive symptom severity, this study’s second aim was to investigate which components of pairs of affective traits (shared versus unique) are related to endothelial function. Finally, given that the mechanisms underlying affective trait-endothelial function relationships in depressed adults are unknown, this study’s third aim was to explore traditional CVD risk status as a candidate mediator of observed relationships. To achieve these aims, I combined pre-treatment, cross-sectional data from three randomized controlled trials involving 138 depressed primary care patients with no history of clinical CVD. Assessments included validated self-report questionnaires for affective traits, brachial artery flow-mediated dilation (FMD) for endothelial function, and 10-year Framingham risk score for traditional CVD risk status. I conducted structural equation modeling (SEM) with confirmatory factor analysis to examine the relationships of interest after adjusting for age, sex, race/ethnicity, education, and baseline arterial diameter. Although the shared variance between each affective trait pair could not be modeled due to poor fit, adequate fitting models revealed that hostility/anger and the unique components of hostility/anger were associated with poorer endothelial function (standardized coefficients = -.18 and -.22, respectively). All of the other affective traits and their components (depression, anxiety, positive affect, unique depression, unique anxiety, and unique positive affect) were not related to endothelial function (all ps > .08). Traditional CVD risk status did not partially explain the relationship between the unique components of hostility/anger and endothelial function (standardized coefficient for the indirect effect = .00; p = .89). If my results are supported by future findings, it would suggest that depressed adults with hostility/anger (a) may be a subgroup of the depressed population at greater risk of developing CVD and (b) may be in need of earlier, more intense, and/or different CVD primary prevention efforts. Future studies are needed to confirm this relationship and identify underlying mechanisms.

Indiana University-Purdue University Indianapolis (IUPUI) / Streptococcus mutans, an inhabitant of oral biofilm or dental plaque, adheres to the tooth surface via protein antigen I/II (PA I/II). Pathologic lesions of atherosclerosis (AT) and infective endocarditis (IE) harbor S. mutans. Serotypes f and k strains with collagen binding protein genes cbm and cnm are uncommon in the mouth, but these are the most prevalent S. mutans strains in AT and IE tissues and can invade endothelial cells (EC) in vitro. Tobacco use increases the risk for cardiovascular and oral diseases. Oral S. mutans encounter many substances including nicotine. Arginine is present in saliva and the EC glycocalyx that coats and protects ECs from shear forces of blood flow. Prior studies demonstrated arginine alters S. mutans biofilm. This work characterizes S. mutans serotype k strains and serotype c strains, the most prevalent in the mouth. The effects of nicotine and arginine on biofilm mass, metabolic activity and EC invasion were investigated. Biofilm production by serotypes c and k strains did not differ; there were no differences in responses to nicotine and arginine between these serotypes. Increased production of biofilm was associated with the cbm and cnm genes. Nicotine increased biofilm for all strains whereas arginine plus nicotine reduced bacteria and the extracellular polymeric substances. Previous EC invasion studies were performed with planktonic cultures of S. mutans; therefore, EC invasion by biofilm was evaluated. Significant factors for EC invasion by S. mutans are presence of the cbm gene and lack of PA I/II expression on the bacterial cell surface. Presence of the cnm gene increased EC invasion by biofilm but not planktonic cells. Planktonic cells of six strains invaded better than biofilm, whereas four strains showed increased invasion by biofilm cells. Neither nicotine nor arginine significantly altered the ability of S. mutans biofilm cells to invade ECs. Not all strains with cbm or cnm and no PA I/II expression invaded EC. A strain with PA I/II expression and without cbm and cnm genes invaded EC. While cbm, cnm and PA I/II expression are predictors of EC invasion, additional mechanisms for EC invasion by S. mutans remain to be revealed. / 2021-08-21

Indiana University-Purdue University Indianapolis (IUPUI) / Fracture repair is costly and difficult to treat. One of the main causations of nonunion is a lack of essential blood supply. The needed blood is supplied by the growth of new blood vessels, a process known as angiogenesis, that invade the damaged tissue early in the healing process. We proposed using bone tissue engineering as an effective therapy. This therapy uses stem cells to aid in tissue regeneration. Endothelial progenitor cells (EPCs) were selected due to their ability to form tube-like networks in vitro. EPCs were isolated from murine bone marrow and lung tissue. We tested EPC’s tube forming, proliferative, and wound migration ability in vitro. To test their ability in vivo we created a femoral fracture in young and old mice. EPCs were seeded to the fracture site upon a collagen scaffold. The in vitro studies displayed that the bone marrow and lung-derived endothelial cells presented EPC traits. In the mouse fracture model bone marrow, endothelial cells did not significantly improve the healing process. In the future, we want to improve our cell extraction and purification method, as well as test a new stem cell delivery biomaterial. We also want to select and use a growth factor (GF) that can help to promote bone regeneration in tandem with the EPCs.

BACKGROUND: Protein disulfide isomerase A3 (PDIA3, also named GRP58, ER-60, ERp57) is conserved across species and mediates protein folding in the endoplasmic reticulum. PDIA3 is, reportedly, a chaperone for STAT3. However, the role of PDIA3 in regulating mitochondrial bioenergetics and STAT3 phosphorylation at serine 727 (S727) has not been described. METHODS: Mitochondrial respiration was compared in immortalized human cerebral microvascular cells (CMEC) wild type or null for PDIA3 and in whole organism C. Elegans WT or null for pdi-3 (worm homologue). Mitochondrial morphology and cell signaling pathways in PDIA3-/- and WT cells were assessed. PDIA3-/- cells were subjected to oxygen-glucose deprivation (OGD) to determine the effects of PDIA3 on cell survival after injury. RESULTS: We show that PDIA3 gene deletion using CRISPR-Cas9 in cultured CMECs leads to an increase in mitochondrial bioenergetic function. In C. elegans, gene deletion or RNAi knockdown of pdi-3 also increased respiratory rates, confirming a conserved role for this gene in regulating mitochondrial bioenergetics. The PDIA3-/- bioenergetic phenotype was reversed by overexpression of WT PDIA3 in cultured PDIA3-/- CMECs. PDIA3-/- and siRNA knockdown caused an increase in phosphorylation of the S727 residue of STAT3, which is known to promote mitochondrial bioenergetic function. Increased respiration in PDIA3-/- CMECs was reversed by a STAT3 inhibitor. In PDIA3-/- CMECs, mitochondrial membrane potential and reactive oxygen species production, but not mitochondrial mass, was increased, suggesting an increased mitochondrial bioenergetic capacity. Finally, PDIA3-/- CMECs were more resistant to oxygen-glucose deprivation, while STAT3 inhibition reduced the protective effect. CONCLUSIONS: We have discovered a novel role for PDIA3 in suppressing mitochondrial bioenergetic function by inhibiting STAT3 S727 phosphorylation.

Chronic inflammation is a complex immune response linked to several diseases. The first step in the inflammatory response is the recruitment of immune cells to the endothelium of the vascular wall. This process is mediated by E/P-Selectin, for which no antagonist efficiently interacts to limit the inflammatory response. Previous work identified transcription factor GATA5 as a key regulator of endothelial homeostasis and revealed an altered expression of inflammatory genes in human endothelial cells with loss of GATA5. The objective of my project is to understand the role of GATA5 in selectin-dependent vascular inflammation and to develop selectin inhibitors. I used biochemical, cellular and in vivo approaches to evaluate a series of novel small molecules for their ability to interfere with selectin binding to their ligand, PSGL-1. The work identified a new lead candidate, LCB 2248, for the development of new E/P-Selectin antagonists and contributed to the understanding of the role of GATA5 in cell recruitment and adhesion. The mechanistic insight gathered and the identification of an E/P-Selectin antagonist will hopefully pave the way for the development of effective treatments for patients with chronic inflammation.

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