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Open Access Publications from the University of California

Scholarly Works (20 results)

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UC Davis Electronic Theses and Dissertations (2021)

Atherosclerosis is a chronic inflammatory disease of arteries that leads to heart attack and stroke in over 3 million people per year in the US. The main risk factors for atherosclerotic cardiovascular disease (ASCVD) include dyslipidemia and disorders of metabolism. Moreover, there is a strong association between arterial flow characteristics and the local regulation of the inflammation that promotes ASCVD. Triglyceride rich lipoproteins (TGRL) are elevated in the circulation of individuals at risk for ASCVD and were shown to act synergistically with low magnitude shear stress (SS) to promote an inflammatory response by cultured human aortic endothelial cells (HAECs). This response was characterized by maximal vascular cell adhesion molecule (VCAM)-1 expression and was dependent upon an endoplasmic reticulum (ER) stress response. The magnitude of this response correlated strongly with enrichment in certain polyunsaturated fatty acid (PUFA) metabolites within the circulating lipoproteins that may be further metabolized by several enzymatic pathways in endothelial cells to produce an array of inflammatory mediators. We hypothesized that shifting endothelial cyclooxygenase (COX)-2-dependent PUFA metabolism alters the ER stress-mediated VCAM 1 upregulation in response to TGRLs.

TGRLs isolated from postprandial human plasma following a high fat test meal were characterized via flow cytometry as pro- or anti-inflammatory based on their impact on up- or down-regulation of VCAM-1 expression by HAEC relative to stimulation with tumor necrosis factor (TNF)α. TNFα stimulated HAEC monolayers were exposed to a SS gradient via artery-on-a-chip (AOC) microfluidics in the presence of TGRL and/or pharmacological intervention to induce or inhibit COX-2. ER stress markers that promote maximal VCAM-1 expression by TGRL in inflamed HAEC were analyzed by western blot. Despite a strong inverse correlation between COX-2 and VCAM-1 expression observed in the SS gradient, the results did not strongly support a direct effect of COX-2 in mediating ER stress-induced VCAM-1 expression but implicated cross talk with other metabolic pathways. COX-2 expression was suppressed by low magnitude (atherosusceptible) SS and enhanced by high magnitude (atheroprotective) SS in the AOC device, registering a 37% increase from static control at 12 dynes/cm2. COX-2 induction with phorbal-12,13-dibutyrate attenuated VCAM-1 across the SS gradient, reducing peak expression at 2 dynes/cm2 by 62%. COX-2 upregulation by pharmacological induction or high magnitude SS suppressed ER stress-mediated VCAM-1 elicited by TNFα and TGRLs. These results are consistent with a protective role for COX- 2 in reducing endothelial inflammation that promotes atherosclerosis and reveal the potential for endothelial PUFA metabolism to modulate an inflammatory response to TGRL. Furthermore, a potent effect of low SS to enhance VCAM-1 expression while suppressing COX-2 expression was observed, consistent with its ability to promote an atherosusceptible phenotype, and demonstrating its importance when studying the inflammation promoting atherosclerosis.

    Cover page: COX-2 Induction Suppresses Endothelial Inflammation Elicited by Lipoproteins and Low Shear Stress
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    UC Davis Previously Published Works (2012)
    Abstract Background The INNER NO OUTER (INO) gene, which encodes a YABBY-type transcription factor, specifies and promotes the growth of the outer integument of the ovule in Arabidopsis. INO expression is limited to the abaxial cell layer of the developing outer integument of the ovule and is regulated by multiple regions of the INO promoter, including POS9, a positive element that when present in quadruplicate can produce low-level expression in the normal INO pattern. Results Significant redundancy in activity between different regions of the INO promoter is demonstrated. For specific regulatory elements, multimerization or the addition of the cauliflower mosaic virus 35S general enhancer was able to activate expression of reporter gene constructs that were otherwise incapable of expression on their own. A new promoter element, POS6, is defined and is shown to include sufficient positive regulatory information to reproduce the endogenous pattern of expression in ovules, but other promoter regions are necessary to fully suppress expression outside of ovules. The full-length INO promoter, but not any of the INO promoter deletions tested, is able to act as an enhancer-blocking insulator to prevent the ectopic activation of expression by the 35S enhancer. Sequence conservation between the promoter regions of Arabidopsis thaliana, Brassica oleracea and Brassica rapa aligns closely with the functional definition of the POS6 and POS9 regions, and with a defined INO minimal promoter. The B. oleracea INO promoter is sufficient to promote a similar pattern and level of reporter gene expression in Arabidopsis to that observed for the Arabidopsis promoter. Conclusions At least two independent regions of the INO promoter contain sufficient regulatory information to direct the specific pattern but not the level of INO gene expression. These regulatory regions act in a partially redundant manner to promote the expression in a specific pattern in the ovule and suppress expression outside of ovules. Establishment of this pattern requires cooperation and competition between multiple positive and negative regulatory elements.
      Cover page: Positive- and negative-acting regulatory elements contribute to the tissue-specific expression of INNER NO OUTER, a YABBY-type transcription factor gene in Arabidopsis
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      • Peer Reviewed
      UC Davis Previously Published Works (2017)
      Atherosclerosis impacts arteries where disturbed blood flow renders the endothelium susceptible to inflammation. Cytokine activation of endothelial cells (EC) upregulates VCAM-1 receptors that target monocyte recruitment to atherosusceptible regions. Endoplasmic reticulum (ER) stress elicits EC dysregulation in metabolic syndrome. We hypothesized that ER plays a central role in mechanosensing of atherosusceptible shear stress (SS) by signaling enhanced inflammation. Aortic EC were stimulated with low-dose TNFα (0.3 ng/ml) in a microfluidic channel that produced a linear SS gradient over a 20mm field ranging from 0-16 dynes/cm2. High-resolution imaging of immunofluorescence along the monolayer provided a continuous spatial metric of EC orientation, markers of ER stress, VCAM-1 and ICAM-1 expression, and monocyte recruitment. VCAM-1 peaked at 2 dynes/cm2 and decreased to below static TNFα-stimulated levels at atheroprotective-SS of 12 dynes/cm2, whereas ICAM-1 rose to a maximum in parallel with SS. ER expansion and activation of the unfolded protein response also peaked at 2 dynes/cm2, where IRF-1-regulated VCAM-1 expression and monocyte recruitment also rose to a maximum. Silencing of PECAM-1 or key ER stress genes abrogated SS regulation of VCAM-1 transcription and monocyte recruitment. We report a novel role for ER stress in mechanoregulation at arterial regions of atherosusceptible-SS inflamed by low-dose TNFα.
        Cover page: Atherosusceptible Shear Stress Activates Endoplasmic Reticulum Stress to Promote Endothelial Inflammation
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        UC Davis Previously Published Works (2014)
        Severely burned patients benefit from intensive insulin therapy (IIT) for tight glycemic control (TGC). The authors evaluated the clinical impact of automatic correction of hematocrit and ascorbic acid interference for bedside glucose monitoring performance in critically ill burn patients. The performance of two point-of-care glucose monitoring systems (GMSs): 1) GMS1, an autocorrecting device, and 2) GMS2, a noncorrecting device were compared. Sixty remnant arterial blood samples were collected in a prospective observational study to evaluate hematocrit and ascorbic acid effects on GMS1 vs GMS2 accuracy paired against a plasma glucose reference. Next, we enrolled 12 patients in a pilot randomized controlled trial. Patients were randomized 1:1 to receive IIT targeting a TGC interval of 111 to 151 mg/dl and guided by either GMS1 or GMS2. GMS bias, mean insulin rate, and glycemic variability were calculated. In the prospective study, GMS1 results were similar to plasma glucose results (mean bias, -0.75 [4.0] mg/dl; n = 60; P = .214). GMS2 results significantly differed from paired plasma glucose results (mean bias, -5.66 [18.7] mg/dl; n = 60; P = .048). Ascorbic acid therapy elicited significant GMS2 performance bias (29.2 [27.2]; P < .001). Randomized controlled trial results reported lower mean bias (P < .001), glycemic variability (P < .05), mean insulin rate (P < .001), and frequency of hypoglycemia (P < .001) in the GMS1 group than in the GMS2 group. Anemia and high-dose ascorbic acid therapy negatively impact GMS accuracy and TGC in burn patients. Automatic correction of confounding factors improves glycemic control. Further studies are warranted to determine outcomes associated with accurate glucose monitoring during IIT.
          Cover page: Clinical Impact of Sample Interference on Intensive Insulin Therapy in Severely Burned Patients
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          UC Davis Previously Published Works (2021)
          Increased expression of vascular cell adhesion molecule (VCAM)-1 on the activated arterial endothelial cell (EC) surface critically contributes to atherosclerosis which may in part be regulated by epigenetic mechanisms. This study investigated whether and how the clinically available histone deacetylases 1 and 2 (HDAC1/2) inhibitor drug Romidepsin epigenetically modulates VCAM-1 expression to suppress atherosclerosis. Methods: VCAM-1 expression was analyzed in primary human aortic EC (HAEC) treated with Romidepsin or transfected with HDAC1/2-targeting siRNA. Methylation of GATA6 promoter region was examined with methylation-specific PCR assay. Enrichment of STAT3 to GATA6 promoter was detected with chromatin immunoprecipitation. Lys685Arg mutation was constructed to block STAT3 acetylation. The potential therapeutic effect of Romidepsin on atherosclerosis was evaluated in Apoe -/- mice fed with a high-fat diet. Results: Romidepsin significantly attenuated TNFα-induced VCAM-1 expression on HAEC surface and monocyte adhesion through simultaneous inhibition of HDAC1/2. This downregulation of VCAM-1 was attributable to reduced expression of transcription factor GATA6. Romidepsin enhanced STAT3 acetylation and its binding to DNA methyltransferase 1 (DNMT1), leading to hypermethylation of the GATA6 promoter CpG-rich region at +140/+255. Blocking STAT3 acetylation at Lys685 disrupted DNMT1-STAT3 interaction, decreased GATA6 promoter methylation, and reversed the suppressive effects of HDAC1/2 inhibition on GATA6 and VCAM-1 expression. Finally, intraperitoneal administration of Romidepsin reduced diet-induced atherosclerotic lesion development in Apoe -/- mice, accompanied by a reduction in GATA6/VCAM-1 expression in the aorta. Conclusions: HDAC1/2 contributes to VCAM-1 expression and atherosclerosis by suppressing STAT3 acetylation-dependent GATA6 promoter methylation. These findings may provide a rationale for HDAC1/2-targeting therapy in atherosclerotic heart disease.
            Cover page: HDAC1 and 2 regulate endothelial VCAM-1 expression and atherogenesis by suppressing methylation of the GATA6 promoter
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            UC Davis Previously Published Works (2019)
            Elevated triglyceride-rich lipoproteins (TGRL) in circulation is a risk factor for atherosclerosis. TGRL from subjects consuming a high saturated fat test meal elicited a variable inflammatory response in TNFα-stimulated endothelial cells (EC) that correlated strongly with the polyunsaturated fatty acid (PUFA) content. This study investigates how the relative abundance of oxygenated metabolites of PUFA, oxylipins, is altered in TGRL postprandially, and how these changes promote endothelial inflammation. Human aortic EC were stimulated with TNFα and treated with TGRL, isolated from subjects' plasma at fasting and 3.5 hrs postprandial to a test meal high in saturated fat. Endothelial VCAM-1 surface expression stimulated by TNFα provided a readout for atherogenic inflammation. Concentrations of esterified and non-esterified fatty acids and oxylipins in TGRL were quantified by mass spectrometry. Dyslipidemic subjects produced TGRL that increased endothelial VCAM-1 expression by ≥35%, and exhibited impaired fasting lipogenesis activity and a shift in soluble epoxide hydrolase and lipoxygenase activity. Pro-atherogenic TGRL were enriched in eicosapentaenoic acid metabolites and depleted in esterified C18-PUFA-derived diols. Abundance of these metabolites was strongly predictive of VCAM-1 expression. We conclude the altered metabolism in dyslipidemic subjects produces TGRL with a unique oxylipin signature that promotes a pro-atherogenic endothelial phenotype.
              Cover page: Oxylipins in triglyceride-rich lipoproteins of dyslipidemic subjects promote endothelial inflammation following a high fat meal
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              • Peer Reviewed
              UC Davis Previously Published Works (2020)

              Aims

              Mammalian target of rapamycin (mTOR) inhibitors used in drug-eluting stents (DES) to control restenosis have been found to delay endothelialization and increase incidence of late-stent thrombosis through mechanisms not completely understood. We revealed that mTOR inhibition (mTORi) upregulated the expression of cell growth suppressor IRF-1 in primary human arterial endothelial cells (HAEC). This study aimed to examine how mTOR-regulated IRF-1 expression contributes to the suppressive effect of mTORi on arterial endothelial proliferation.

              Methods and results

              Western blotting, quantitative PCR, and a dual-luciferase reporter assay indicated that mTOR inhibitors rapamycin and torin 1 upregulated IRF-1 expression and increased its transcriptional activity. IRF-1 in turn contributed to the suppressive effect of mTORi by mediating HAEC apoptosis and cell cycle arrest in part through upregulation of caspase 1 and downregulation of cyclin D3, as revealed by CCK-8 assay, Annexin V binding assay, measurement of activated caspase 3, BrdU incorporation assay, and matrigel tube formation assay. In a mouse model of femoral artery wire injury, administration of rapamycin inhibited EC recovery, an effect alleviated by EC deficiency of IRF-1. Chromatin immunoprecipitation assay with HAEC and rescue expression of wild type or dominant-negative IRF-1 in EC isolated from Irf1-/- mice confirmed transcriptional regulation of IRF-1 on the expression of CASP1 and CCND3. Furthermore, mTORi activated multiple PKC members, among which PKCζ was responsible for the growth-inhibitory effect on HAEC. Activated PKCζ increased IRF1 transcription through JAK/STAT-1 and NF-κB signaling. Finally, overexpression of wild type or mutant raptor incapable of binding mTOR indicated that mTOR-free raptor contributed to PKCζ activation in mTOR-inhibited HAEC.

              Conclusions

              The study reveals an IRF-1-mediated mechanism that contributes to the suppressive effects of mTORi on HAEC proliferation. Further study may facilitate the development of effective strategies to reduce the side effects of DES used in coronary interventions.
                Cover page: IRF-1 mediates the suppressive effects of mTOR inhibition on arterial endothelium
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                UC Davis Previously Published Works (2019)
                Epoxyeicosatrienoic acids (EETs) are formed from the metabolism of arachidonic acid by cytochrome P450s. EETs promote angiogenesis linked to tumor growth in various cancer models that is attenuated in vivo by cyclooxygenase 2 (COX-2) inhibitors. This study further defines a role for COX-2 in mediating endothelial EET metabolism promoting angiogenesis. Using human aortic endothelial cells (HAECs), we quantified 8,9-EET-induced tube formation and cell migration as indicators of angiogenic potential in the presence and absence of a COX-2 inducer [phorbol 12,13-dibutyrate (PDBu)]. The angiogenic response to 8,9-EET in the presence of PDBu was 3-fold that elicited by 8,9-EET stabilized with a soluble epoxide hydrolase inhibitor (t-TUCB). Contributing to this response was the COX-2 metabolite of 8,9-EET, the 11-hydroxy-8,9-EET (8,9,11-EHET), which exogenously enhanced angiogenic responses in HAECs at levels comparable to those elicited by vascular endothelial growth factor (VEGF). In contrast, the 15-hydroxy-8,9-EET isomer was also formed but inactive. The 8,9,11-EHET also promoted expression of the VEGF family of tyrosine kinase receptors. These results indicate that 8,9-EET-stimulated angiogenesis is enhanced by COX-2 metabolism in the endothelium through the formation of 8,9,11-EHET. This alternative pathway for the metabolism of 8,9-EET may be particularly important in regulating angiogenesis under circumstances in which COX-2 is induced, such as in cancer tumor growth and inflammation.
                  Cover page: Epoxyeicosatrienoic acid (EET)-stimulated angiogenesis is mediated by epoxy hydroxyeicosatrienoic acids (EHETs) formed from COX-2
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                  UC Irvine Previously Published Works (2011)
                    Cover page: Chronic myeloid leukemia 2011: successes, challenges, and strategies--proceedings of the 5th annual BCR-ABL1 positive and BCR-ABL1 negative myeloproliferative neoplasms workshop.
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                    UC Davis Previously Published Works (2023)
                    Human induced pluripotent stem cells (hiPSCs) not only provide an abundant source of vascular cells for potential therapeutic applications in vascular disease but also constitute an excellent model for understanding the mechanisms that regulate the differentiation and the functionality of vascular cells. Here, we reported that myocyte enhancer factor 2C (MEF2C) transcription factor, but not any other members of the MEF2 family, was robustly upregulated during the differentiation of vascular progenitors and endothelial cells (ECs) from hiPSCs. Vascular endothelial growth factors (VEGF) strongly induced MEF2C expression in endothelial lineage cells. The specific upregulation of MEF2C during the commitment of endothelial lineage was dependent on the extracellular signal regulated kinase (ERK). Moreover, knockdown of MEF2C with shRNA in hiPSCs did not affect the differentiation of ECs from these hiPSCs, but greatly reduced the migration and tube formation capacity of the hiPSC-derived ECs. Through a chromatin immunoprecipitation-sequencing, genome-wide RNA-sequencing, quantitative RT-PCR, and immunostaining analyses of the hiPSC-derived endothelial lineage cells with MEF2C inhibition or knockdown compared to control hiPSC-derived ECs, we identified TNF-related apoptosis inducing ligand (TRAIL) and transmembrane protein 100 (TMEM100) as novel targets of MEF2C. This study demonstrates an important role for MEF2C in regulating human EC functions and highlights MEF2C and its downstream effectors as potential targets to treat vascular malfunction-associated diseases.
                      Cover page: Role of MEF2C in the Endothelial Cells Derived from Human Induced Pluripotent Stem Cells
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