bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 189: The Role of Spleen Tyrosine Kinase, Syk, in CD36-Toll Like Receptor Cooperative Signaling in Atherosclerosis
Authors: Dutta Ray Tathagat , Ramkhelawon Bhama , Moore Kathryn J
Published: July 2021

Atherosclerosis is characterized by chronic sterile inflammation of the artery wall in which cells of the monocyte lineage accumulate in response to the deposition of low density lipoprotein (LDL). We previously established that recognition of oxidized LDL (oxLDL) by CD36 triggers assembly of a novel Toll-like receptor heterodimer composed of TLR4 and TLR6. Here we set out to understand the molecular mechanisms of CD36/TLR4/TLR6 activation and establish how it triggers downstream signals that lead to the expression of the pro-inflammatory mediators that have been directly implicated in the deleterious effects of oxLDL and atherosclerosis progression. By confocal microscopy we demonstrate that oxLDL induces CD36, TLR4 and TLR6 co-localization in intracellular compartments, but not on the cell surface of macrophages. Notably, inhibition of oxLDL endocytosis (with Dynasore) or lysosomal maturation (with Bafilomycin A or NH4Cl) blocks CD36-TLR4-TLR6 complex formation and oxLDL-induced cytokine responses in macrophages. These data indicate that both ligand internalization and lysosomal acidification are required for assembly of a functional CD36/TLR4/TLR6 signaling complex. Notably, CD36 contains a hemi-ITIM motif in the C-terminus that is reported to interact with the spleen tyrosine kinase Syk through its SH2 domain. As Syk has recently been implicated in the trafficking of CD14 and TLR4 to the endosome in response to LPS, we investigated the role of this kinase in CD36/TLR4/TLR6 signaling. We find that Syk is required for CD36 internalization and TLR4/TLR6 heterodimerization. Using a pharmacological inhibitor, we show that inhibition of Syk activity blocks oxLDL-induced TLR4-TLR6 co-precipitation and abrogates macrophage expression of both MyD88- (IL-1b, CXCL1) and TRIF-dependent (CCL5) cytokines/chemokines. Together, our data are consistent with a key role for Syk in the trafficking of CD36 and oxLDL to the lysosome, where it coordinates the assembly of a functional TLR4-TLR6 heterodimer to initiate signaling. This model highlights the importance of CD36 as a co-receptor that orchestrates TLR4-TLR6 trafficking and assembly to initiate the detrimental inflammatory responses that promote the progression of atherosclerosis.
Abstract 20: Tribbles1 (Trib1) is a Novel Regulator of in vivo Hepatic Fatty Acid Lipogenesis in the Mouse.
Authors: Bauer Robert C , Cui Jian , Kent Anthony P , Rader Daniel J
Published: July 2021

Tribbles1 (TRIB1) was recently identified in genome-wide association studies as being strongly linked to plasma levels of VLDL, HDL, LDL, and TG as well as coronary artery disease in humans. Previous experiments in mice using AAV-mediated hepatic overexpression of Trib1 confirmed this association, as mice overexpressing Trib1 exhibited reductions of 45% and 57% in plasma total cholesterol (TC) and TG, respectively (
Burkhardt et al, 2010
). Here we report a Trib1 liver-specific knockout mouse (Trib1_LSKO) created through AAV-mediated delivery of Cre recombinase into adult mice with a floxed version of Trib1. Four weeks after infection, Trib1_LSKO mice exhibited 21% and 70% increases in TC and TG, respectively (
p
=0.01 and 0.02), as compared to floxed Trib1 littermates infected with null virus (Controls). Trib1_LSKO animals also exhibited a 25% increase in liver weight (
p
<0.01), and histological analysis revealed steatotic livers in LSKO mice. Real-time PCR analysis revealed >2-fold increases in the hepatic transcription of genes involved in fatty acid synthesis in Trib1_LSKO mice as compared to Controls. Examination of hepatic lipids revealed a 78% increase in hepatic TG content (
p
<0.001) of Trib1_LSKO mouse livers, while no significant change in hepatic cholesterol was observed. When de novo lipogenesis was measured using [3H]-acetate, Trib1_LSKO animals exhibited significantly increased production of TG (3.6-fold,
p
<0.001), fatty acids (2.2-fold,
p
=0.02), diacylglycerol (1.8-fold,
p
<0.01), and phospholipids (2-fold,
p
=0.05). Microarray analysis of Trib1_LSKO livers compared to Controls revealed greater than 1,600 genes that were significantly altered between the two groups (fold change>1.5, FDR<10%). Pathway analysis suggested that the altered gene set was enriched for genes downstream of C/EBP and C/EBP. Western blot analysis of liver extracts showed increases in both C/EBP and C/EBP levels in Trib1_LSKO mice compared to Controls. In conclusion, Trib1 is a novel regulator of de novo lipogenesis in mice, presumably through the regulation of lipogenic gene transcription. This transcriptional control may be regulated by increased levels of C/EBP and/or C/EBP, or an as yet undetermined target of Trib1.
Abstract 251: Effects of n-3 Fatty Acids Supplementation on Endothelial Function and Inflammation in Peripheral Artery Disease: The OMEGA-PAD Trial
Authors: Grenon Marlene , Owens Christopher , Alley Hugh , Chong Karen , Yen Priscilla , Vittinghoff Eric , Harris William , Hughes-Fulford Millie , Rapp Joseph , Conte Michael
Published: July 2021

Background

Patients with peripheral artery disease (PAD) have an increased risk for cardiovascular events, likely related to inflammation and impaired endothelial function. n-3 polyunsaturated fatty acids (n3-PUFAs) have been shown to improve endothelial function and reduce inflammation in other cohorts. We hypothesized that n3-PUFAs would improve endothelial function and the inflammatory profile in patients with PAD.

Methods

This is a randomized, double-blind, placebo-controlled trial: the OMEGA-PAD trial (
NCT01310270
). Eighty patients aged 50 and more with intermitted claudication and an ankle-brachial index (ABI) of <0.9 presenting to vascular surgery clinic at the Veterans Affairs Medical Center in San Francisco will be randomized to n3-PUFAs 2.2 g orally twice daily (total of 4.4gm/day) or a matched placebo for 1 month. Outcome measurements are done at baseline and after 1 month. The primary endpoint is a change in endothelial function measured by brachial artery flow mediated, endothelium-dependent vasodilation (FMD). Secondary endpoints include a change in inflammatory markers (C-reactive protein, IL-6, sICAM-1 and TNF-α), improvement in lipid profile (LDL, triglycerides, HDL), blood pressure and walking distance by questionnaire. The omega-3 index will be measured to ensure physiological treatment effect.

Results

Recruitment for the OMEGA-PAD trial started in April 2011. Fifty male veterans have been enrolled and randomized. The mean age is 67 ± 9 years. Mean index limb ABI is 0.8 ± 0.2. CAD is present in 36%, hypertension in 92%, and diabetes mellitus in 32%. 47% are current smokers and 45% are former smokers. Mean HgA1c is 8±1, LDL 93±37 mg/dL, triglycerides 160±98 mg/dL and HDL 43 ±12 mg/dL. Baseline brachial FMD is 7±4% indicating an overall impairment of endothelial function in this cohort. The inflammatory burden of this cohort is substantial as evidenced by hsCRP 5±5 mg/L. The baseline omega-3 index is 5±2%.

Conclusions

The OMEGA-PAD trial will test the novel hypothesis that n3-PUFAs supplementation improves functional and inflammatory parameters in a cohort at high vascular risk. The results of this study will provide valuable mechanistic insight into PUFAs as well as inform on this class of agents for cardiovascular outcome trials.
Abstract 255: Cytoprotective Effects of Erythropoietin and Erythropoietin-derivatives in Ischaemic Human Myotubes and the Role of Pi3k/akt Signalling Pathway
Authors: Yu Rebekah Sian Hwee , Baker Daryll , Abraham David , Tsui Janice
Published: July 2021

Objectives

Erythropoietin (Epo) has tissue-protective effects in response to injury, acting through the EpoR-βcR heteroreceptor. We have previously demonstrated the presence and interaction of the EpoR and βcR in human skeletal muscle. Here we aim to investigate the potential cytoprotective effects of Epo and an Epo-derivative (ARA-290) in a human in vitro model of skeletal muscle and establish a potential downstream signalling pathway utilised in protecting cells from apoptosis (including Jak-2, PI3k/Akt, NFkB).

Methods

Gastrocnemius muscle biopsies were obtained from patients with critical limb ischaemia and control samples were obtained from non-ischaemic patients. Human myoblasts were isolated from muscle biopsies, cultured, and allowed to differentiate into myotubes in order to investigate the cytoprotective effects of Epo and ARA-290 on myotubes subjected to simulated ischaemia. The PI3k inhibitors, LY294002 and wortmannin, were then used to determine the role of PI3k/Akt pathway in mediating cytoprotection. Following this, inhibitors against the upstreatm (Jak-2) and downstream (NFkB) molecules were also investigated. Western blot analysis, using the pro-apoptotic marker cleaved caspase-3 was performed and compared with levels of Akt and phosphorylated-Akt, using western blot analysis.

Results

Exogenous administration of Epo and ARA-290 were able to ameliorate the ischaemia-induced apoptosis on isolated human myotubes as shown by a significant reduction in cleaved caspase-3 expression. Addition of all inhibitors, to ARA-290 or Epo pre-treated cells, abolished the reduction in apoptosis.

Conclusion

The ability of ARA-290 to attenuate apoptosis in human myotubes undergoing ischaemic insult suggests a potential role in tissue protection in skeletal muscle injury. We propose that the PI3k/Akt signalling pathway is involved in mediating this cytoprotection.
Abstract 266: The Usefulness of a Collagen Plug Vascular Closure Device in Patients Undergoing Coronary and Peripheral Percutaneous Intervention
Authors: Owens John T , Tordini Andrea , Bhatty Shaun , Danyi Peter , Patel Kalpesh , Jovin Ion S
Published: July 2021

Background

Vascular access site complications can follow coronary and peripheral angioplasty procedures and be associated with significant morbidity. We compared the complication rates of the Angioseal closure device with complication rates after manual compression in patients undergoing coronary and peripheral interventional procedures.

Methods and Results

We studied 553 predominantly male patients undergoing percutaneous coronary, peripheral interventions, FFR or IVUS at a Veterans Administration Medical Center. The mean age was 64.6 years. Angioseal closure devices were used in 388 (70%) patients and manual compression was used in 165 (30%) patients. Major groin complications such as arteriovenous fistula, pseudoaneurysm or hematoma greater than 5 cm occurred in 16 (4.16%) patients who had an Angioseal closure device and 11 (6.67%) patients in whom manual compression was used for hemostasis (odds ratio [OR] 0.63, 95% confidence interval [CI] 0.29-1.40; P=0.26). Other complications such as minor bleeding requiring additional manual compression, bruising, pain and swelling occurred in 32 (8.42%) patients who had an Angioseal closure device and 21 (12.4%) patients who had manual compression (OR 0.65, 95% CI 0.36-1.17; P=0.15). After adjustment for other comorbid factors such as age, type of anticoagulant used, peripheral vascular disease and chronic kidney disease, the association of major complications with the use of the Angioseal closure device remained similar to manual compression (OR 0.75, 95% CI 0.28-1.91; P=0.49). However, there was a trend for fewer minor complications with the closure device (OR 0.57, 95% CI 0.26-1.08; P=0.08).

Conclusion

In this cohort of predominantly male patients undergoing percutaneous coronary and peripheral intervention, the use of the Angioseal closure device was not associated with a decreased risk of major groin complications, but was associated with a trend for a decreased risk of minor complications compared to manual compression.
Abstract 269: Development of Acute Heart Failure Following Cardiac Catheterization in Patient’s With Pre-Existing Peripheral Artery Disease
Authors: Harris Gregory , Patwardhan Manish , Cameron Yara , Adusumalli Sundeep , Daggubati Ramesh
Published: July 2021

Background

Peripheral artery disease has long been considered a CHD risk equivalent resulting in increased CAD, angina, CHF, and MI. Regardless, it is not known if PAD results in increased likelihood of acute complications following cardiac catheterization.

Methods

The medical records of 3817 patients undergoing cardiac catheterization at a university medical center were reviewed. Development of acute heart failure 72 hours after the procedure was compared to pre-existing medical conditions including peripheral artery disease using multiple logistic regression analysis. Results in the table below are reported as odds ratios with 95% confidence interval. Chi-square was measured comparing PAD with post procedure heart failure. Relative risk reported with 95% confidence interval.

Results

As shown below, there was a statistically significant difference between development of acute heart failure up to 72 hours after cardiac catheterization in patients with pre-existing PAD as well as history of hyperlipidemia. Chi-square demonstrated 22% of patients who developed heart failure also had pre-existing PAD (χ
2
(1,N=3816)=6.336,
p
=0.012). Relative risk of developing heart failure with pre-existing PAD was 1.782 (95% CI 1.13-2.80).

Conclusion

We have demonstrated in this retrospective analysis of 3817 patients, an increased risk of developing heart failure in patients with pre-existing PAD and dyslipidemia following cardiac catheterization. These results further justify optimal lipid management as an integral component of cardiovascular health, not only in relation to chronic disease, but also in the presence of acute injury.

Legend

PAD = Peripheral Artery Disease
Abstract 277: Downregulation of PDGF-D is Associated with Increased Collagen Production in Abdominal Aortic Aneurysm
Authors: Vorkapic Emina , Mäyränpää Mikko , Roy Joy , Eriksson Per , Wågsäter Dick
Published: July 2021

BACKGROUND

Abdominal aortic aneurysm (AAA) is characterized by loss of aortic smooth muscle cells (aoSMCs) and remodeling of extracellular matrix, such as collagen. Platelet-derived growth factor-D (PDGF-D) is the latest discovered member of the PDGF/VEGF family and involved in proliferation and migration of SMCs. Here we analyzed the expression of PDGF-D in human and murine AAA. Furthermore, we studied the potential role of PDGF-D in regulation of collagen in cultured aoSMC.

METHOD AND RESULTS

Gene expression analysis of abdominal aortas revealed a significant lower expression of PDGF-D (-85%) and its receptor PDGFR-β (-73%) in human aneurysmal wall compared to non-aneurysmal control aortas. PDGF-D and PDGFR-β was mainly localized to SMC and endothelial cells as determined by immunohistochemical analysis and was expressed weaker in aneurysmal samples compared with controls. In mice, luminal aortic diameter was increased by 60% (P<0.001) in aortas treated with 0.5M CaCl
2
compared to saline treated control aortas. In agreement with human AAA, PDGF-D expression was significantly decreased in mice with AAA (-60%) compared to controls. A decrease in the expression of PDGF-D was strongly correlated to both α-actin (r=0.95, P<0.001) and SM22α (r=0.94, P<0.001) expression. A decreased expression of PDGF-D (r=-0.76, P<0.05) and α-actin (r=-0.88, P<0.01) was correlated with increased expression of the apoptosis marker caspase-3. To investigate the potential inhibitory role of PDGF-D in collagen remodeling, cultured aoSMCs were stimulated with 20 ng/ml of human recombinant PDGF-D or PBS as control for 24 and 48 hours. Treatment of aoSMC with rh-PDGF-D resulted in a decrease in the synthesis of collagen-III (P=0.023) and -IV (P=0.01) as well as prolyl 4-hydroxylase (P=0.01) and lysyl oxidase (P=0.02) after 48 hours. The synthesis of collagen-I was unaffected with increased PDGF-D.

CONCLUSION

Our findings imply that PDGF-D functions as a negative regulator of collagen synthesis and that lack of PDGF-D could therefore be important for the AAA process.
Abstract 312: Mapping the Inflammatory Macrophage Response during Arteriovenous Fistula Maturation by In Vivo Molecular Imaging
Authors: Cui Jie , Kessinger Chase W , McCarthy Jason R , Lin Charles , Jaffer Farouc A
Published: July 2021

Introduction

Inflammation plays a critical role in neointimal hyperplasia (NH), which leads to arteriovenous fistula (AVF) failure in hemodialysis patients. However, the spatial distribution of macrophages in vivo post AVF creation remains unclear. In this study, we mapped the distribution of macrophages in AVF using intravital fluorescence microscopy (IVFM) and a fluorescence macrophage nanosensor, CLIO-VT680. We hypothesized that the intensity of CLIO-VT680 signals would illuminate the topography of AVF inflammation.

Methods

AVF was created in C57BL/6J mice (n=5) by end to side anastomosis between the jugular vein and the ipsilateral carotid artery. Mice were injected with CLIO-VT680 (10mg/kg) at day 13 post AVF creation and imaged by IVFM 24 hours later. AVF was resected at week 6, and Von Gieson (VVG) staining was performed. Mean CLIO signal intensity (MSI) was measured every 60μm from the anastomosis. Target to background ratios (TBRs) were calculated as the MSI of AVF divided by the MSI of the control artery. TBR ratio was calculated by the TBRs at different distance away from the anastomosis divided by the TBR at the anastomotic site.

Results

The survival rate of mice after AVF creation was 100%. The penetration depth of IVFM was 200μm. IVFM detected significantly higher TBRs of CLIO signals near the anastomotic site (p<0.05). There is a linear relationship between TBR ratios and the distance away from the anastomosis (R2=0.99). VVG staining of resected AVF showed the volume of NH decreased as the distance away from the anastomosis increased.

Conclusion

Macrophages response can be detected via CLIO-VT680 using IVFM. In vivo molecular imaging may be able to predict AVF failure.
Abstract 32: Hypercholesterolemia-induced Priming of Hematopoietic Stem and Progenitor Cells Aggravates Atherosclerosis
Authors: Seijkens Tom , Hoeksema Marten A. , Beckers Linda , Meiler Svenja , Smeets Esther , Levels Han , de Winther Menno P.J. , Lutgens Esther
Published: July 2021

During homeostasis hematopoietic stem and progenitor stem cells (HSPCs) give rise to lymphoid and myeloid cells as well as platelets and erythrocytes. However, during chronic inflammatory conditions hematopoiesis is often skewed towards the myeloid lineage, thereby potentially aggravating the ongoing inflammation. Here we investigated the effects of hypercholesterolemia on HSPCs during atherogenesis.

Hypercholesterolemia increased HSPCs, defined as Lin
-
Sca1
+
cKit
-
, in the bone marrow (BM) of LDLr
-/-
mice by 253.1%. The number of granulocyte-monocyte progenitors, BM granulocytes and BM monocytes was increased by 18.1%, 34.8% and 13.2%, respectively. In accordance, the myeloid colony forming potential of hypercholesterolemic BM was increased by 25.8%. Peripheral blood monocytes and granulocytes were increased by 203.0% and 161.1%, respectively.

Competitive bone marrow transplantations (cBMT) in which we compared the effects of normo- vs. hypercholesterolemia primed HSPCs confirmed that the hypercholesterolemic microenvironment activates HSPCs, as reflected by a 26.5% increased reconstitution of peripheral blood leukocytes 10 weeks after the cBMT. Moreover, hypercholesterolemia-primed, and not normocholesterolemia-primed HSPCs acquired an enhanced propensity to generate myeloid cells, especially granulocytes and Ly6C
high
monocytes, even under long-term normocholesterolemic conditions in the recipient animals. cBMT demonstrated that hypercholesterolemia-induced activation of HSPCs increased atherosclerosis in LDLr
-/-
mice by 122.1% and increased CD45.1
+
plaque leukocytes by 76.1%. Macrophages differentiated from hypercholesterolemia-primed BM produced increased levels of TNFα (+21.3%), IL6 (+17.4%) and MCP1 (+10.5%) compared to their normocholesterolemic counterparts, demonstrating that hypercholesterolemia-induced priming of HSPCs increased the inflammatory phenotype of their mature offspring.

These results demonstrate that hypercholesterolemia-induced priming of HSPCs aggravates atherosclerosis by skewing hematopoiesis towards the pro-inflammatory myeloid lineages. Inhibition of this pro-inflammatory differentiation pathway on HSPC level has the potential to reduce atherosclerosis.
Abstract 321: The E12/id3 Axis Regulates Ccr6 Expression In B Cells Which Are Associated With Human Atherosclerosis
Authors: Morris-Rosenfeld Sam , Perry Heather , Lipinski Michael , McSkimming Chantel , Garmey James , Taylor Angela , McNamara Coleen
Published: July 2021

Background

Aortic homing of B cells and B cell mediated atheroprotection depend on the helix-loop-helix (HLH) transcription factor Id3. We identified that CCR6, a downstream target of Id3, is also essential for B cell homing to the aorta. Id3 functions through dominant negative inhibition of basic-HLH transcription factors such as E proteins. We have shown that a non-synonomous single nucleotide polymorphism in Id3 correlates with human carotid intimal medial thickness (cIMT). The protein encoded by this risk allele shows attenuated interaction with E12 yet whether this property is shared with other E proteins and the potential impact of Id3 and E12 on CCR6 expression in B cell subsets is unknown.

Results

Co-IPs of ancestral and polymorphic Id3 with the four known E proteins showed that E12, and not other E proteins, had significantly decreased interaction with polymorphic Id3. To address the regulatory role of Id3 on CCR6 in B cells, B cell subsetting by flow cytometry was done showing that B cell specific Id3 knockout mice (Id3fl/flApoe-/-CD19cre/+) indeed have significantly fewer CCR6 positive B cells which is in agreement with our previous findings in global Id3 knockout mice. To address the regulatory role of E12 on CCR6 expression similar subsetting of global E12 knockout mice (E12-/-Apoe-/-) was done showing they have significantly more CCR6 positive B cells of all subsets (B1a, B1b, B2) compared to wildtype. In particular the atheroprotective B1a subset had the greatest percentage increase due to loss of E12 compared to other subsets. Finally, we addressed whether CCR6 on human B cells correlates with human atherosclerosis. We found that coronary plaque burden and stenosis as measured by intravascular ultrasound (IVUS) in patients undergoing coronary catheterization correlated inversely with the average amount of CCR6 on B cells.

Conclusions

Our data suggests that Id3 is a key activator of CCR6 expression in B cells through dominant negative inhibition of E12. Furthermore, it raises the interesting possibility that CCR6 could play an important atheroprotective role through its regulation of B cell homing in humans.
Abstract 324: Tumor Necrosis Factor-a (TNF-a) Mediates Microvascular Dysfunction Induced by High-fat Diet and Murine Cytomegalovirus (mCMV) Infection in a Mouse Model
Authors: Khoretonenko Mikhail V , Stokes Karen Y
Published: July 2021

Cardiovascular diseases are mediated by a variety of risk factors, e.g. obesity and infections. Complicated obesity and mCMV each induce microvascular dysfunction in mouse models. However, the impact of mild obesity on microvascular homeostasis and its possible synergism with cytomegalovirus infection remain unclear.

We have shown that 8 wks high-fat diet (HFD) increases leukocyte recruitment in cremasteric post-capillary venules, and mCMV accelerates and sustains this response. We hypothesized that HFD and/or mCMV impair phenylephrine-mediated vasoconstriction in arterioles, and that the arteriolar and venular responses are mediated by an imbalance between TNF-α and leptin, vs. adiponectin.

Mice were inoculated with mock or mCMV and at 4 wks post-infection (pi) started on HFD or left on normal diet (ND). At 12 wks pi, leukocyte recruitment and arteriolar vasoconstriction were assessed by intravital microscopy. Adiponectin and leptin were measured by ELISA. Some mice were treated with anti-TNF-α antibodies 2 days prior to observation.

mCMV infection alone did not alter any parameter when compared to Mock-ND. Arteriolar vasoconstriction to phenylephrine was impaired in both HFD groups vs. Mock-ND mice. TNF-α inhibition partially restored this impaired vasoconstriction. Leukocyte adhesion was increased in both HFD groups vs. ND mice, and was ameliorated by anti-TNF-α antibody treatment in mCMV-HFD, but not Mock-HFD mice. Adiponectin levels in HFD vs. ND groups were slightly increased, and leptin levels were ~5-6-fold higher.

Our results suggest that HFD-/+mCMV infection impairs arteriolar vasoconstriction via a TNF-α dependent mechanism. TNF-α independent HFD-induced leukocyte adhesion in venules is changed to a TNF-α dependent pathway by mCMV infection. Increased leptin levels in HFD groups correlate with previous findings linking obesity and increased leptin. Increased adiponectin levels in HFD animals are contrary to responses previously reported for complicated obesity and may represent an initial compensatory role for adiponectin in the response to HFD-induced weight gain and inflammation. However, the roles for leptin and adiponectin require future investigation.
Abstract 33: Cardiac Fibroblasts Reprogram Into Endothelial Like Cells in a p53 Dependent Manner After Acute Ischemic Cardiac Injury
Authors: Ubil Eric , Deb Arjun
Published: July 2021

The mammalian heart exhibits poor regenerative capacity after acute ischemic injury and heals primarily by fibrosis. Recently, several groups have demonstrated that cardiac fibroblasts can be reprogrammed to adopt myogenic fates using exogenous transcription factors. However, the ability of cardiac fibroblasts to adopt specific cellular fates in the absence of exogenous factors is unclear. Here, we demonstrate that a subset of cardiac fibroblasts adopt endothelial characteristics after ischemic cardiac injury in the absence of any added factors. Using mice harboring genetically labeled fibroblasts (Col1a2CreERT:R26RTdTomato), we show that 34 +/- 3% (mean, SEM) of labeled cardiac fibroblasts in the injury border zone express endothelial markers such as VE-cadherin. Fibroblast derived endothelial cells comprised 25 +/- 2% of total and 8 +/- 2% of luminal endothelial cells at the border zone 3 days after injury. To better understand fibroblast-endothelial reprogramming we subjected cardiac fibroblasts to cellular stress (serum starvation) and found that they formed tubes on Matrigel and up-regulated endothelial specific genes (e.g. VE-cadherin, Flk1, Flt1) 6-20 fold. We show that reprogramming of fibroblasts to endothelial like cells ex vivo is p53 dependent. Inhibiting p53 activity by pharmacological means (Pifithrin-α) or genetic deletion in fibroblasts (Col1a2CreERT:p53fl/fl) led to a 94% decrease in Matrigel tube formation and 90% reduction in endothelial gene expression. Moreover, we observed that p53 levels in cardiac fibroblasts were more than 10-fold higher at the injury border zone using semi-quantitative immunofluorescent staining. Injection of a p53 activator after injury doubled p53 levels in cardiac fibroblasts and increased the rate of fibroblast-endothelial reprogramming by 43%. Enhanced fibroblast-endothelial reprogramming was also associated with decreased collagen deposition 3 days post injury. In summary, we show that cardiac fibroblasts are able to adopt endothelial cell like fates both in vivo and ex vivo in a p53 dependent manner. Manipulation of fibroblast to endothelial reprogramming could represent a novel therapeutic strategy to increase post infarct angiogenesis and enhance function in the injured heart.
Abstract 338: Patient with Triple Threat Survives DVT and Pulmonary Embolism Complication
Authors: Shalshin Aleksander , Mensch Alan , Kapoor Karnika
Published: July 2021

Introduction

The risk factors for DVT and complication of PE are firmly documented in literature. Virchow’s triad describes the three most common categories responsible for venous thrombosis occurrence: Hypercoagulation, Stasis and Endothelial Injury.

Our patient was diagnosed with near fatal episode of DVT and PE as a result of all 3 risk categories in combination, based on iatrogenic and inherited causes.

Discussion

Patient presented with left leg edema and shortness of breath. He described recent arthroscopic surgery performed on left knee for meniscal tear. Listed home medications included testosterone supplement for muscle building purposes. Furthermore, patient’s family history provided vague details about possible history of blood clots on maternal side.

Patient physical examination pertinent demonstrated left lower extremity edema and tenderness on palpation.

Imaging - CT scan showed multiple PE, saddle PE and lower extremity Doppler sonogram provided evidence of DVT in left femoral and popliteal vein.

Further evaluation into hypercoagulation disorders showed that patient had an inherited condition of Protein C deficiency.

Our patient’s etiology for occurrence of DVT and PE was identified with utilization of history, physical examination, imaging and laboratory investigation.

The unique combination of iatrogenic (surgery and testosterone use) and non-iatrogenic (inherited) risk factors resulting in this patient’s clinical diagnosis are an important reminder to clinicians of all specialties to consider various elements of Virchow’s triad when approached with problem of venous thrombosis. Our patient demonstrates a classic element of each - endothelial injury (recent knee surgery), stasis (limited weight bearing post operatively), hypercoagulation (testosterone use and Protein C deficiency). Economic burden of DVT, PE remains high and is often associated with post thrombotic complications mounting economic and clinical expenditure. Timely detection, appropriate therapy and provider education about high risk population has the potential for clinical and economic benefit.

Our patient was placed on anticoagulation, counseled about testosterone use, and Protein C deficiency condition.
Abstract 380: Aging Enhances Endoplasmic Reticulum Stress-Induced Apoptosis in Murine Macrophages
Authors: Goldstein Daniel R , Song Yang
Published: July 2021

Introduction and hypothesis

Aging enhances atherosclerosis for unclear reasons. As macrophage apoptosis and endoplasmic reticulum (ER) stress contribute to atherosclerosis, we examined if aging sensitizes these cells to apoptosis during ER stress.

Methods and Results

Peritoneal macrophages were isolated from young (aged 2-4 months) and aged (aged 16-18 months) mice, exposed to the ER stress inducer tunicamycin (TM) in vitro, and apoptosis was measured by Annexin V staining via fluorescent microscopy. We found that aged macrophages exhibited significantly more apoptosis than young macrophages (see Figure). We next measured key ER stress proteins in macrophages by Western blot to determine the underlying molecular pathways impacted by aging. With aging, we found reduced activation of inositol-requiring enzyme-1 (IRE1α), a key ER stress transducer. We next examined if augmenting activated IRE1α levels in aged macrophages reduced apoptosis during ER stress. We employed siRNA to knock down x-box binding protein 1 (XBP1), a downstream effector of IRE1α, which has been shown to induce feedback activation of IRE1α in hepatocytes. siRNA to XBP1 significantly reduced tunicamycin-induced cell apoptosis in aged macrophages from 26.1±0.408% to 5.48±1.38% (p<0.05) but not in young macrophages.

Conclusions

Our study has uncovered a novel, age-dependent interaction by which macrophages undergo apoptosis upon ER stress, and suggests that enhancing IRE1α activation will alleviate aging-augmented ER stress and subsequent apoptosis. This novel interaction may have important implications for the pathogenesis of atherosclerosis with aging.
Abstract 381: IL-19 Inhibits Atherosclerosis in LDLR-/- Mice by Modulating Cholesterol Uptake in Macrophages and Vascular Smooth Muscle Cells
Authors: Gabunia Khatuna , Ellison Stephen P , Richards James M , Kelemen Sheri E , Autieri Michael V
Published: July 2021

IL-19 is a recently described, putative anti-inflammatory cytokine which had previously been ascribed to be leukocyte specific. IL-19 is not detected in normal artery, but we detected IL-19 in multiple cell types in human atherosclerotic plaque suggesting a role for this interleukin in atherosclerosis. The purpose of this study was to determine whether administration of exogenous IL-19 could attenuate development of pre-formed atherosclerotic plaque, and to identify potential molecular mechanisms. LDLR-/- mice were fed high-fat diet for 12 weeks and then administered with 10ng/g/day IL-19 or PBS for an additional 8 weeks. En face analysis demonstrated that IL-19 could halt, but not reverse existing plaque (26.7+/-1.7%, 41.03+/-3.1%, 23.70+/-2.6% for baseline, PBS control, and IL-19-treated mice). Foam cell formation by macrophages and vascular smooth muscle cells (VSMC) is a hallmark event during atherosclerosis. Nothing has been reported regarding IL-19 effects on macrophage or VSMC lipid uptake; we therefore investigated whether IL-19 affects macrophage and VSMC cholesterol handling. Addition of IL-19 to wild-type bone marrow derived macrophages (BMDM) significantly promoted oxLDL uptake, conversely, BMDM from IL-19-/- mice had significantly less oxLDL uptake compared to wild-type BMDM. Addition of IL-19 to wild type BMDM significantly increased expression of scavenger receptor B1 (SR-B1), and decreased expression of inflammatory cytokines TNFα, IL-12b, MCP1. Interestingly, converse results were obtained with VSMC, as addition of IL-19 to wild-type VSMC decreased uptake of oxLDL (
p<0.05
) and decreased expression of scavenger receptor CD36. VSMC isolated from IL-19-/- mice had increased uptake of oxLDL (p<0.0001). It is reported that M2 macrophages participate in plaque regression. IL-19 decreased IL-12b and significantly promoted the polarization of anti-inflammatory M2 phenotype in BMDM as evidenced by the increased expression of YM1 and IL-10 mRNA. These data demonstrate that IL-19 can inhibit progression of existing atherosclerotic plaque by modulating lipid metabolism in VSMC and macrophages and by promoting macrophage differentiation into an alternative, anti-inflammatory M2 phenotype.
Abstract 382: Anti-MAA Antibodies in Sprague Dawley Rats are Increased in Response to a High Fat Western Diet
Authors: Duryee Michael J , Dusad Anand , Shurmur Scott W , Johnston Michael D , Garvin Robert P , Hunter Carlos D , Anderson Daniel R , Thiele Geoffrey M
Published: July 2021

Introduction

Malondialdehyde/Acetaldehyde (MAA) modified proteins have been suggested to play a role in the development/progression of atherosclerosis. Circulating antibodies directed against these proteins have recently been shown to be associated with the severity of the disease. More specifically, the isotype of the antibody to MAA correlated with either an acute MI (IgG) or stable plaque formation (IgA) formation. MAA is thought to form as a result of the oxidation of fat(s) and thus the concentration and antibody response should reflect the amount of fat in the diet.

Objective

The purpose of this study was to evaluate the antibody responses to MAA modified proteins following immunization and high fat western diet feeding in rats.

Methods

Male Sprague Dawley rats were immunized with MAA-modified protein weekly for 5 weeks and then assayed for antibodies to these proteins. Animals were then separated into the following groups: chow sham, chow MAA immunized, high fat sham, and high fat MAA immunized. The high fat animals were fed a Western diet with 2-thiouracil for 12 weeks, bled every 3 weeks, and serum assayed for the presence of circulating MAA antibodies.

Results

Prior to feeding with high fat diet, rats immunized with MAA-modified protein had a significant increase (P<0.001) in serum antibodies directed against these modified proteins compared to controls (N of 4 per group). Following feeding of high fat diet antibody concentrations increased 6 fold in the high fat MAA immunized group compared to the chow MAA immunized group (P<0.05). Antibodies in the high fat sham and chow sham had only minimal increases in antibodies to these proteins.

Conclusions

These data demonstrate that following immunization with MAA-modified proteins, circulating antibodies are produced that increase following consumption of a high fat Western diet. It suggests that MAA-modified proteins are produced at low levels following normal diet, producing antibodies which act as a normal clearance method for altered protein. When high fat consumption increases these antibody levels are increased in response to the oxidative stress.

Implications

Use of these antibodies as a biomarker in the future may help predict the onset or progression of atherosclerosis.
Abstract 39: The Transient Receptor-Potential Channel 6 Regulates Thromboxane A2 Receptor-Operated Calcium Entry and Plays a Critical Role in Platelet Function
Authors: Khasawneh Fadi T , Espinosa Enma V , Lin Olivia A , Murad John P
Published: July 2021

Although changes in the intracellular levels of calcium is a central step in platelet activation, the underlying mechanism of changes that are dependent on receptor-operated calcium entry (ROCE) remains to be defined. Furthermore, it was proposed, though never proven, that the Transient Receptor-Potential Channels (TRPCs) may play a key role in this process. In this connection, we have previously shown that TRPC6 plays a vital role in physiological hemostasis and thrombogenesis. However, the underlying mechanism by which TRPC6 modulates these processes also remains to be determined. Based on these considerations, we hypothesized that TRPC6 plays an essential role in ROCE and hence platelet function. Using a (genetic) TRPC6 knockout (KO) mouse approach, our preliminary studies revealed that these platelets exhibited a defect in platelet aggregation induced by the thromboxane A
2
receptor (TPR) agonist
U46619.
Conversely, the aggregation response triggered by ADP or the thrombin receptor activating peptide 4 (TRAP4) was found to be comparable to that of the wild-type (WT) platelets. Separate studies revealed that the TRPC6 deficient platelets were also found to exhibit a defect in TPR-mediated dense granule (ATP) secretion, whereas that of ADP and TRAP4 was normal. Moreover, we observed a defect in integrin GP IIb-IIIa activation that was again specific to TPR (normal activation in response to ADP and TRAP4), suggesting a defect in inside-out signaling. Finally, TPR-dependent CE was also found to be deficient in the TRPC6 KO platelets, unlike that stimulated by ADP or TRAP4. Future studies will further investigate the molecular mechanism of TRPC6-regulated platelet function and CE. Taken together, these findings demonstrate for the first time that TRPC6 regulates CE in a TPR-dependent manner and that this regulation consequently modulates platelet aggregation, secretion, as well as GP IIb-IIIa activation. These studies may define a new therapeutic target for managing multiple thrombosis-based disorders.
Abstract 424: Mechanisms of Restenosis in Type 2 Diabetes Mellitus
Authors: Wang Hongfeng , Kersten Judy R , Toth Jeffrey M , LaDisa John F
Published: July 2021

Stents are widely employed to decrease cardiovascular risk in patients with type 2 diabetes mellitus (T2DM); however, restenosis limits their success. Mechanisms responsible for restenosis in T2DM are incompletely elucidated; however, low wall shear stress (WSS) and altered intracellular signaling likely contribute. We tested the hypothesis that neointimal hyperplasia (NH) after bare-metal stenting in T2DM is due to vascular remodeling (enhanced formation of advanced glycation end-products (AGEs), increased downstream vascular resistance (DVR), and decreased WSS) in T2DM; and that decreasing AGEs with ALT-711 (Alagebrium) mitigates this response.

Stents were implanted into the abdominal aorta of Zucker lean (ZL), obese (ZO), and diabetic (ZD) rats. After 21 days, blood flow and pressure data were recorded; and the stented region was sectioned for NH quantification or casted and imaged for WSS distributions by computational fluid dynamics modeling. Arterial segments (thoracic and abdominal aorta, carotid, iliac, femoral and arterioles) were harvested to detect AGEs related collagen cross-linking, and protein expression including TGFβ and receptor for AGE (RAGE).

DVR was elevated, whereas, WSS was significantly decreased in ZD compared to ZL and ZO rats, respectively (14.5±1.9 vs 30.6±1.6 and 25.4±2.2dyn/cm
2
; mean±SEM p<0.05). Intra-strut thickness (NH) was increased in ZO but not ZD rats. ALT-711 reduced DVR in ZD rats (15.6±2.5x10
5
to 8.39±0.6x10
5
dyn·s/cm
5
), decreased NH (ZL: 7.7±1.0 to 4.3±0.9%; ZO: 12.0±1.5 to 4.9±0.8%; ZD: 9.4±0.7 to 3.7±0.4%) and restored WSS in all rats. Increases in AGEs related collagen cross-linking were present in the arterioles of ZD rats, but reduced by treatment with ALT-711. No differences in RAGE or TGFβ expression were observed in treated compared to untreated rats.

In conclusion, ALT-711 decreased AGEs related collagen cross-linking, DVR, and normalized intra-stent WSS in rats with T2DM. These findings suggest that vascular remodeling may play an important role in modulating restenosis. Although TGFβ and RAGE expression did not appear to be modified by ALT-711, other intracellular signaling pathways that influence restenosis during T2DM remain to be explored.
Abstract 439: Niacin Does Not Accelerate Reverse Cholesterol Transport in Man
Authors: Dunbar Richard L , Cuchel Marina , Millar John S , Baer Amanda , Poria Rahul , Freifelder Richard H , Pryma Daniel A , Billheimer Jeffrey J , Rader Daniel J
Published: July 2021

Background

HDL may be atheroprotective by accelerating reverse cholesterol transport (RCT). Niacin raises HDL cholesterol (HDL-c) and niacin monotherapy up to 3g/d prevented coronary events, possibly by accelerating RCT. However, up to 2g/d niacin did not prevent outcomes beyond aggressive LDL lowering including statins, arguing against RCT-mediated atheroprotection. We developed a novel method to assess macrophage-to-lipoprotein RCT in vivo. Preliminary studies in mice demonstrate that albumin-bound tritiated cholesterol (3H-Chol) is rapidly removed ( 95% in 30min) by resident macrophages. We monitored subsequent reappearance over 8d by macrophage-to-lipoprotein RCT as 3H-Chol rate of appearance (RA pre-post therapy, %3H-Chol injected/mol cholesterol/h) and tracer fecal excretion (%3H-Chol injected/g feces), and further analyzed RCT by multi-compartmental modeling.

Results

Niacin raised HDL-c 44% vs 17% on placebo (p=0.02) and dropped non HDL-c 40% (p<0.0001). Niacin and placebo did not differ by HDL 3H-Chol RA (placebo: 4.16-3.72, niacin: 3.24-2.61, p=0.8), total plasma 3H-Chol RA (placebo: 2.70-2.42, niacin: 2.35-2.14, p=0.9), macrophage-to-HDL transfer rate (placebo: 4.8-4.2, niacin: 5.1-4.7 fractional transfer/h, p=0.7), ex vivo cholesterol efflux capacity ratio (placebo: 0.73-0.82, niacin: 0.91-0.95, p=0.2), or tracer fecal excretion (placebo: 34.1-37.4, niacin: 21.2-28.0, p=0.6).

Conclusion

Despite potently raising HDL-c, high-dose niacin had no benefit on in vivo or ex vivo macrophage-to-lipoprotein RCT or fecal excretion in man, suggesting niacin’s atheroprotective potential lies elsewhere.
Abstract 477: HDL Inhibition of Murine Atherosclerosis Lesion Development is Dependent on Strain Background
Authors: Sontag Timothy J , Krishack Paulette A , Biswas Tapan K , Getz Godfrey S , Reardon Catherine A
Published: July 2021

Inbred mouse strains vary greatly in their relative atherosensitivity, with C57BL/6 (C57) being the most sensitive and hence the preferred strain for most atherosclerosis studies. Among the resistant strains, FVBN/J (FVB) has been relatively well studied. On the ApoE knockout (E°) background, both mouse strains develop atherosclerotic lesions which are exacerbated by feeding a high fat/high cholesterol western-type diet (WTD). However, the FVB-E° mouse exhibits nearly 10-fold smaller lesions than the C57-E°. Additionally, FVB mice have plasma HDL cholesterol levels that are twice as high as C57. Wild type FVB mice have been shown to have lower inflammatory cell recruitment to the peritoneum upon thioglycollate treatment than C57. Our lab has generated ApoE/ApoA-I double knockout mice (EA°, lacking HDL) on both C57 and FVB background to compare the effect of HDL deficiency on lesion development. We hypothesize that the loss of HDL will have a greater effect on the more lesion susceptible C57 than the resistant FVB.

8 week C57 and FVB E° and EA° mice were kept on a WTD for 6 or 10 weeks and lesions were quantified in the innominate artery, ascending aorta, and aortic root. In all cases mice in the C57 background displayed significantly greater lesion size than mice in the FVB background, except in the ascending aorta of the E° mice at the earlier time point, despite higher plasma cholesterol levels in the FVB mice. The presence of ApoA-I was protective (E° vs EA°) in the C57 strain in the ascending aorta at both time points and its protection became significant in the innominate artery at the 10 week time point. No additional protection was afforded by ApoA-I in the FVB strain. ApoA-I deficiency in FVB mice did not affect inflammatory cell recruitment to the peritoneal cavity or plasma markers of LDL oxidation as greatly as in C57 mice. Plasma monocyte counts both before and after 6 weeks on WTD were higher in C57 mice with no effect of ApoA-I in either strain.

In conclusion, the FVB strain is less sensitive than C57 to lesion development at 3 major arterial sites with no protection afforded by HDL/ApoA-I, whereas HDL/ApoA-I is significantly protective in the C57 strain. This protection may result from the anti-inflammatory/anti-oxidative nature of C57 HDL that is not observed in FVB mice.
Abstract 490: Acute Superior Mesenteric Artery Occlusion Presenting as Strangulated Inguinal Hernia
Authors: Cao Shanjin , Huo Lihong , Ge Rongrong , Sun Li , Carrey Zev , Petrillo Richard
Published: July 2021

An 85yo woman presented with diffused colicky abdominal pain and vomiting for 12hr. Patient still had bowel movement and passed flatus. Patient was otherwise healthy without past medical or surgical history, and taking no medications. PE: no distress, afebrile, vitals are stable. Cardiopulmonary exam only revealed tachycardia; Abdomen: soft, non-tender, no distension, bowel sounds hyperactive; there was an 8[[Unable to Display Character: ]]6 cm irreducible, firm but non-tender right inguinal hernia. Labs: leukocytosis, bicarbonate 20. CT abdomen with oral contrast suggested small bowel obstruction secondary to right inguinal hernia. The hernia was then reduced manually. Post the reduction patient had one foul smell watery bowel movement.

However, patient deteriorated quickly. Repeat labs: bicarbonate 8, anion gap 23, lactic acid 11, ABG: 7.01/29/246/7/99%. EKG: A-fib with tachycardia. Patient’s blood pressure dropped necessitating IV fluid and vasopressor support. Strangulated hernia with septic shock was suspected and emergency laparotomy was performed. Ascending, transverse and descending colon past the splenic curve were found necroses with foul-smelling turbid fluid intraperitoneally; the small bowel was minimally dilated with a kink 25cm beyond the ileocelcal valve with evidence of previous incarceration. Subtotal colectomy and partial small bowel resection with ileostomy was performed. Pathology confirmed terminal ileum and colonic gangrene; recent mesentery venous thrombi. Despite appropriate therapy, patient continued to be in septic shock and developed multiple organ failure, a stroke, and then brain death.

Acute mesenteric ischemia (AMI) is a rare abdominal emergency with high mortality, which could be caused by direct strangulation of the SMA by the inguinal hernia (Tiwary SK et al 2008) or thromoboembolism, e.g., emboli from A-fib, septic emboli, local thrombosis. Since there is no evidence that the incarcerated portion of small bowel had necrosis, this case is mostly likely caused by the paroxysmal A-fib-derived emboli. Due to the lack of typical clinical expression, a high index of suspicion is essential for prompt diagnosis. CT angiography is the current cornerstones for diagnosis of AMI, so treatment can be initiated expeditiously.