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1.  Shear Stress-Activated Wnt-Angiopoietin-2 Signaling Recapitulated Vascular Repair in Zebrafish Embryos 
Fluid shear stress intimately regulates vasculogenesis and endothelial homeostasis. The canonical Wnt/β-catenin signaling pathways play an important role in differentiation and proliferation. In this study, we investigated whether shear stress activated Angiopoietin-2 (Ang-2) via the canonical Wnt signaling pathway with an implication in vascular endothelial repair.
Approach and Results
Oscillatory shear stress(OSS) up-regulated both TOPflash Wnt reporter activities and the expression of Ang-2 RNA and protein in human aortic endothelial cells (HAEC) accompanied by an increase in nuclear β-catenin intensity. OSS-induced Ang-2 and Axin-2 mRNA expression was down-regulated in the presence of a Wnt inhibitor, IWR-1, but was up-regulated in the presence of a Wnt agonist, LiCl. Ang-2 expression was further down-regulated in response to a Wnt signaling inhibitor, DKK-1, but was up-regulated by Wnt agonist Wnt3a. Both DKK-1 and Ang-2 siRNA inhibited endothelial cell migration and tube formation, which were rescued by human recombinant Ang-2. Both Ang-2 and Axin-2 mRNA down-regulation was recapitulated in the heat-shock inducible transgenic Tg (hsp70l:dkk1-GFP) zebrafish embryos at 72 hours post fertilization (hpf). Ang-2 morpholino injection of Tg (kdrl:GFP) fish impaired subintestinal vessel (SIV) formation at 72hpf, which was rescued by zebrafish Ang-2 mRNA (zAng-2) co-injection. Inhibition of Wnt signaling with IWR-1 also down-regulated Ang-2 and Axin-2 expression, and impaired vascular repair after tail amputation, which was rescued by zAng-2 injection.
Shear stress activated Ang-2 via canonical Wnt signaling in vascular endothelial cells, and Wnt-Ang-2 signaling is recapitulated in zebrafish embryos with a translational implication in vascular development and repair.
PMCID: PMC4169303  PMID: 25147335
Angiopoietin-2; Wnt signaling; endothelial repairs; human aortic endothelial cells; zebrafish; DKK-1/Dickkopfs-1; vasculogenesis
2.  Imaging Body Fat: Techniques and Cardiometabolic Implications 
Obesity is a world-wide epidemic and is associated with multiple comorbidities. The mechanisms underlying the relationship between obesity and adverse health outcomes remain poorly understood. This may be due to several factors including the crude measures used to estimate adiposity, the striking heterogeneity between adipose tissue depots, and the influence of fat accumulation in multiple organs. In order to advance our understanding of fat stores and associated co-morbidities in humans, it will be necessary to image adiposity throughout the body and ultimately also assess its functionality. Large clinical studies are demonstrating the prognostic importance of adipose tissue imaging. Newer techniques capable of imaging fat metabolism and other functions of adipose tissue may provide additional prognostic utility and may be useful in guiding therapeutic interventions.
PMCID: PMC4169325  PMID: 25147343
obesity; fat composition; adipose tissue imaging
3.  Shear stress-initiated signaling and its regulation of endothelial function 
Atherosclerosis develops preferentially at branches and curvatures of the arterial tree, where blood flow pattern is disturbed rather than being laminar, and wall shear stress has an irregular distribution without defined directions. The endothelium in the atherosusceptible regions, in comparison to that in atheroresistant regions, shows activation of pro-proliferative and pro-inflammatory gene expressions, reduced production of nitric oxide (NO), increased leukocyte adhesion and permeability, as well as other atheroprone phenotypes. Differences in gene expressions and cell phenotypes have been detected in endothelia residing in native atherosusceptible and atheroresistant regions of the arteries, or in arteries from animal models with artificial creation of disturbed flow. Similar results have also been shown in in vitro systems that apply controlled shear stresses with or without clear directions to cultured endothelial cells (ECs) in fluid-dynamically designed flow-loading devices. The available evidence indicates that the coordination of multiple signaling networks, rather than individual separate pathways, link the mechanical signals to specific genetic circuitries in orchestrating the mechanoresponsive networks to evoke comprehensive genetic and functional responses.
PMCID: PMC4169328  PMID: 24876354
Shear stress; Mechanotransduction; Endothelial phenotype; Atherogenesis
4.  Role of flow-sensitive microRNAs in endothelial dysfunction and atherosclerosis – “Mechanosensitive Athero-miRs” 
Atherosclerosis preferentially occurs in arterial regions exposed to disturbed flow, in part, due to alterations in gene expression. MicroRNAs (miRNAs) are small, noncoding genes that post-transcriptionally regulate gene expression by targeting messenger RNA transcripts. Emerging evidence indicates that alteration of flow conditions regulate expression of miRNAs in endothelial cells both in vitro and in vivo. These flow-sensitive microRNAs, known as “mechano-miRs”, regulate endothelial gene expression, and can regulate endothelial dysfunction and atherosclerosis. MiRNAs such as, miR-10a, -19a, -23b, -17~92, -21, -663, -92a, -143/145, -101, -126, -712, 205, and -155, have been identified as mechano-miRs. Many of these miRNAs were initially identified as flow-sensitive in vitro and were later found to play a critical role in endothelial function and/or atherosclerosis in vivo through either gain-of-function or loss-of-function approaches. The key signaling pathways that are targeted by these mechano-miRs include the endothelial cell cycle, inflammation, apoptosis, and nitric oxide signaling. Furthermore, we have recently shown that the miR-712/205 family, which is upregulated by disturbed flow, contributes to endothelial inflammation and vascular hyper-permeability by targeting tissue inhibitor of metalloproteinase-3 (TIMP3), which regulates metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs). The mechano-miRs that are implicated in atherosclerosis are termed as “mechanosensitive athero-miRs” and are potential therapeutic targets to prevent or treat atherosclerosis. This review summarizes the current knowledge of mechanosensitive athero-miRs and their role in vascular biology and atherosclerosis.
PMCID: PMC4169332  PMID: 25012134
microRNAs; atherosclerosis; endothelial dysfunction; mechanosensitive athero-miR; shear stress; disturbed flow; MMPs; ADAMs; TIMP3
5.  Hypertriglyceridemia associated with the c.553G>T APOA5 SNP results from aberrant hetero-disulfide bond formation 
Apolipoprotein (apo) A-V is a low abundance plasma protein that modulates triacylglycerol (TG) homeostasis. Gene transfer studies were undertaken in apoa5 (−/−) mice to define the mechanism underlying the correlation between the single nucleotide polymorphism (SNP) c.553G>T in APOA5 and hypertriglyceridemia (HTG).
Approach and Results
Adeno-associated virus (AAV) 2/8 mediated gene transfer of wild type (WT) apoA-V induced a dramatic lowering of plasma TG in apoa5 (−/−) mice while AAV2/8-Gly162Cys apoA-V (corresponding to the c.553G>T SNP: rs2075291) had a modest effect. Characterization studies revealed that plasma levels of WT- and G162C apoA-V in transduced mice were similar and within the physiological range. Fractionation of plasma from mice transduced with AAV2/8-G162C apoA-V indicated that, unlike WT apoA-V, >50% of G162C apoA-V was recovered in the lipoprotein-free fraction. Non-reducing SDS-PAGE immunoblot analysis provided evidence that G162C apoA-V present in the lipoprotein-free fraction, but not that portion associated with lipoproteins, displayed altered electrophoretic mobility consistent with disulfide-linked hetero-dimer formation. Immunoprecipitation followed by liquid chromatography/mass spectrometry of human plasma from subjects homozygous for WT APOA5 and c.553G>T APOA5 revealed that G162C apoA-V forms adducts with extraneous plasma proteins including fibronectin, kininogen-1 and others.
Substitution of Cys for Gly at position 162 of mature apoA-V introduces a free cysteine that forms disulfide bonds with plasma proteins such that its lipoprotein binding and TG modulation functions are compromised.
PMCID: PMC4178935  PMID: 25127531
apolipoprotein A-V; triacylglycerol; lipoprotein; gene therapy; single nucleotide polymorphism
6.  RUC-4: A Novel αIIbβ3 Antagonist for Pre-hospital Therapy of Myocardial Infarction 
Treatment of myocardial infarction (MI) within the first 1–2 hours with a thrombolytic agent, percutaneous coronary intervention, or an αIIbβ3 antagonist decreases mortality and the later development of heart failure. We previously reported on a novel small molecule αIIbβ3 antagonist, RUC-2, that has a unique mechanism of action. We have now developed a more potent and more soluble congener of RUC-2, RUC-4, designed to be easily administered intramuscularly (IM) by autoinjector to facilitate its use in the pre-hospital setting. Here we report the properties of RUC-4 and the antiplatelet and antithrombotic effects of RUC-2 and RUC-4 in animal models.
Approach and Results
RUC-4 was ~20% more potent than RUC-2 in inhibiting human ADP-induced platelet aggregation and much more soluble in aqueous solutions (60–80 mg/ml). It shared RUC-2’s specificity for αIIbβ3 vs αVβ3, did not prime the receptor to bind fibrinogen, or induce changes in β3 identified by a conformation-specific monoclonal antibody. Both RUC-2 and RUC-4 prevented FeCl3-induced thrombotic occlusion of the carotid artery in mice and decreased microvascular thrombi in response to laser injury produced by human platelets infused into transgenic mice containing a mutated von Willebrand factor that reacts with human, but not mouse platelets. IM injection of RUC-4 in non-human primates at 1.9 and 3.85 mg/kg led to complete inhibition of platelet aggregation within 15 minutes, with dose-dependent return of platelet aggregation after 4.5–24 hours.
RUC-4 has favorable biochemical, pharmacokinetic, pharmacodynamic, antithrombotic, and solubility properties as a pre-hospital therapy of MI, but the possibility of increased bleeding with therapeutic doses remains to be evaluated.
PMCID: PMC4180209  PMID: 25147334
αIIbβ3; platelet; myocardial infarction
7.  Recent Highlights of ATVB: Arterial Smooth Muscle 
PMCID: PMC4197844  PMID: 25231633
animal model cardiovascular disease; atherosclerosis; microRNA; neointima; smooth muscle cell; smooth muscle progenitor cell
8.  Rising Like the Phoenix? 
PMCID: PMC4198940  PMID: 25231635
9.  Obstructive Sleep Apnea is Associated with Future Subclinical Carotid Artery Disease: Thirteen-Year Follow-up from the Wisconsin Sleep Cohort 
To determine the longitudinal associations between obstructive sleep apnea (OSA), carotid artery intima-media thickness (IMT), and plaque.
Approach and Results
This is a population-based, prospective cohort study conducted from July, 1989 to November, 2012 on 790 randomly selected employed Wisconsin residents who completed a mean of 3.5 (range 1–6) polysomnograms during the study period. OSA was characterized by the apnea-hypopnea index (AHI, events/hour). Common carotid artery IMT and plaque were assessed by B-mode ultrasound. The mean (SD) time from the first polysomnograms to carotid ultrasound was 13.5 (3.6) years. Multivariable regression models were created to estimate the independent associations of baseline and cumulative OSA exposure with subsequent carotid IMT and plaque. At baseline, participants were mean 47.6 (7.7) years old (55% male, 97% white). AHI was 4.4 (9.0) events/hour (range, 0–97); 7% had AHI>15 events/hour. Carotid IMT was 0.755 (0.161) mm; 63% had plaque. Adjusting for age, sex, body-mass index, systolic blood pressure, smoking, and use of lipid-lowering, antihypertensive, and diabetes medications, baseline AHI independently predicted future carotid IMT (β=0.027 mm/unit log10[AHI+1], p=0.049), plaque presence (odds ratio 1.55 [95% confidence intervals 1.02–2.35], p=0.041) and plaque score (odds ratio 1.30 [1.05–1.61], p=0.018). In cumulative risk factor-adjusted models, AHI independently predicted future carotid plaque presence (p=0.012) and score (p=0.039), but not IMT (p=0.608).
Prevalent OSA is independently associated with increased carotid IMT and plaque over a decade later, indicating increased future cardiovascular disease risk.
PMCID: PMC4259877  PMID: 25189572
Atherosclerosis; Carotid arteries; Epidemiology; Sleep apnea; Ultrasound; Sleep disorders
10.  Pericyte Regulation of Vascular Remodeling Through the CXC Receptor 3 
To understand the role, if any, played by pericytes in the regulation of newly formed vessels during angiogenesis. In this study, we investigate whether pericytes regulate the number of nascent endothelial tubes.
Approach and Results
Using an in vitro angiogenesis assay (Matrigel assay), we demonstrate that pericytes can inhibit vessel formation and induce vessel dissociation via CXCR3-induced involution of the endothelial cells. In a coculture Matrigel assay for cord formation, pericytes prevented endothelial cord formation of human dermal microvascular endothelial cells but not umbilical vein endothelial cells. Blockade of endothelial CXCR3 function or expression inhibited the repressing effect of the pericytes. We further show that pericytes are also able to induce regression of newly formed microvascular cords through CXCR3 activation of calpain. When CXCR3 function was inhibited by a neutralizing antibody or downregulated by siRNA, cord regression mediated by pericytes was abolished.
We show for the first time that pericytes regulate angiogenic vessel formation, and that this is mediated through CXCR3 expressed on endothelial cells. This suggests a role for pericytes in the pruning of immature vessels overproduced during wound repair.
PMCID: PMC4591000  PMID: 24135023
angiogenesis; CXC chemokine receptor 3; endothelial cells; pericytes; wound healing
11.  Humanizing the Problem of Transplant Vasculopathy 
PMCID: PMC4578289  PMID: 22258896
immune system; transplantation
12.  Cholesterol-Induced Membrane Microvesicles As Novel Carriers of Damage–Associated Molecular Patterns Mechanisms of Formation, Action, and Detoxification 
Cholesterol enrichment occurs in vivo when phagocytes ingest retained and aggregated lipoproteins, damaged or senescent cells, and related debris. We previously reported that enrichment of human monocyte/macrophages with unesterified cholesterol (UC) triggers the release of highly procoagulant microvesicles ([MVs], also called microparticles) through induction of apoptosis. We determined whether UC-induced MVs (UCMVs) might transmit endogenous danger signals and, if so, what molecular processes might be responsible for their production, recognition, and detoxification.
Methods and Results
Injection of UCMVs into rats provoked extensive leukocyte rolling and adherence to postcapillary venules in vivo. Likewise, exposure of mouse aortic explants or cultured human endothelial cells to UCMVs augmented the adhesion of human monocytes by several fold and increased endothelial cell intercellular adhesion molecule-1 via nuclear factor-κB activation. To explore molecular mechanisms, we found that UC enrichment of human monocytes, in the absence of other stimuli, induced mitochondrial complex II–dependent accumulation of superoxide and peroxides. A subset of these moieties was exported on UCMVs and mediated endothelial activation. Strikingly, aortic explants from mice lacking lectin–like oxidized low–density lipoprotein receptor-1, a pattern-recognition receptor, were essentially unable to respond to UCMVs, whereas simultaneously treated explants from wild-type mice responded robustly by increasing monocyte recruitment. Moreover, high-density lipoprotein and its associated enzyme paraoxonase-1 exerted unexpected roles in the detoxification of UCMVs.
Overall, our study implicates MVs from cholesterol–loaded human cells as novel carriers of danger signals. By promoting maladaptive immunologic and thrombotic responses, these particles may contribute to atherothrombosis and other conditions in vivo.
PMCID: PMC4568119  PMID: 22814745
microvesicles; microparticles; damage–associated molecular patterns; cholesterol; monocytes; endothelium
13.  Cholesterol Enrichment of Human Monocyte/Macrophages Induces Surface Exposure of Phosphatidylserine and the Release of Biologically-Active Tissue Factor–Positive Microvesicles 
Biologically significant amounts of two procoagulant molecules, phosphatidylserine (PS) and tissue factor (TF), are transported by monocyte/macrophage-derived microvesicles (MVs). Because cellular cholesterol accumulation is an important feature of atherosclerotic vascular disease, we now examined effects of cholesterol enrichment on MV release from human monocytes and macrophages.
Methods and Results
Cholesterol enrichment of human THP-1 monocytes, alone or in combination with lipopolysaccharide (LPS), tripled their total MV generation, as quantified by flow cytometry based on particle size and PS exposure. The subset of these MVs that were also TF-positive was likewise increased by cellular cholesterol enrichment, and these TF-positive MVs exhibited a striking 10-fold increase in procoagulant activity. Moreover, cholesterol enrichment of primary human monocyte-derived macrophages also increased their total as well as TF-positive MV release, and these TF-positive MVs exhibited a similar 10-fold increase in procoagulant activity. To explore the mechanisms of enhanced MV release, we found that cholesterol enrichment of monocytes caused PS exposure on the cell surface by as early as 2 hours and genomic DNA fragmentation in a minority of cells by 20 hours. Addition of a caspase inhibitor at the beginning of these incubations blunted both cholesterol-induced apoptosis and MV release.
Cholesterol enrichment of human monocyte/macrophages induces the generation of highly biologically active, PS-positive MVs, at least in part through induction of apoptosis. Cholesterol-induced monocyte/macrophage MVs, both TF-positive and TF-negative, may be novel contributors to atherothrombosis.
PMCID: PMC4568121  PMID: 17158353
macrophages; microparticles; tissue factor; apoptosis; atherosclerosis
14.  Reconstituted high density lipoprotein therapies - a cause for optimism 
PMCID: PMC4560112  PMID: 25142879
reconstituted HDL; CSL112; cholesterol efflux; reverse cholesterol transport; acute coronary syndrome
15.  Dietary Cholesterol Promotes Adipocyte Hypertrophy and Adipose Tissue Inflammation in Visceral, But Not Subcutaneous, Fat in Monkeys 
Excessive caloric intake is associated with obesity and adipose tissue dysfunction. However, the role of dietary cholesterol in this process is unknown. The aim of this study was to determine whether increasing dietary cholesterol intake alters adipose tissue cholesterol content, adipocyte size, and endocrine function in nonhuman primates.
Approach and Results
Age-matched, male African Green monkeys (n=5 per group) were assigned to one of three diets containing 0.002 (Lo), 0.2 (Med) or 0.4 (Hi) mg cholesterol/Kcal. After 10 weeks of diet feeding, animals were euthanized for adipose tissue, liver, and plasma collection. With increasing dietary cholesterol, free cholesterol (FC) content and adipocyte size increased in a step-wise manner in visceral, but not subcutaneous fat, with a significant association between visceral adipocyte size and FC content (r2=0.298; n=15; p=0.035). In visceral fat, dietary cholesterol intake was associated with: 1) increased pro-inflammatory gene expression and macrophage recruitment, 2) decreased expression of genes involved in cholesterol biosynthesis and lipoprotein uptake, and 3) increased expression of proteins involved in FC efflux.
Increasing dietary cholesterol selectively increases visceral fat adipocyte size, FC and macrophage content, and proinflammatory gene expression in nonhuman primates. Visceral fat cells appear to compensate for increased dietary cholesterol by limiting cholesterol uptake/synthesis and increasing FC efflux pathways.
PMCID: PMC4140949  PMID: 24969772
adipocyte; cholesterol; dietary cholesterol; white adipose tissue; inflammation; African Green monkey; nonhuman primate
16.  Myeloid Cell-Specific ABCA1 Deletion Has Minimal Impact on Atherogenesis in Atherogenic Diet-Fed LDL Receptor Knockout Mice 
Transplantation studies suggest that bone marrow (BM) cell ABCA1 protects against atherosclerosis development. However, the in vivo impact of macrophage ABCA1 expression on atherogenesis is not fully understood because BM contains other leukocytes and hematopoietic stem and progenitor cells. Myeloid-specific ABCA1 knockout (MSKO) mice in the LDL receptor knockout (LDLrKO) C57BL/6 background were developed to address this question.
Approach and Results
Chow-fed MSKO/LDLrKO (DKO) vs. LDLrKO (SKO) mice had similar plasma lipid concentrations, but atherogenic diet (AD)-fed DKO mice had reduced plasma VLDL/LDL concentrations resulting from decreased hepatic VLDL triglyceride secretion. Resident peritoneal macrophages from AD-fed DKO vs. SKO mice had significantly higher cholesterol content, but similar proinflammatory gene expression. Atherosclerosis extent was similar between genotypes after 10–16 wks of AD, but increased modestly in DKO mice by 24 wks of AD. Lesional macrophage content was similar, likely due to higher monocyte flux through aortic root lesions in DKO vs. SKO mice. After transplantation of DKO or SKO BM into SKO mice and 16 wk of AD feeding, atherosclerosis extent was similar and plasma apoB lipoproteins was reduced in mice receiving DKO BM. When differences in plasma VLDL/LDL concentrations were minimized by maintaining mice on chow for 24 wks, DKO mice had modest, but significantly more, atherosclerosis compared to SKO mice.
Myeloid cell ABCA1 increases hepatic VLDL triglyceride secretion and plasma VLDL/LDL concentrations in AD-fed LDLrKO mice, offsetting its atheroprotective role in decreasing macrophage cholesterol content, resulting in minimal increase in atherosclerosis.
PMCID: PMC4140956  PMID: 24833800
cardiovascular disease; atherosclerosis; lipids; lipoproteins; cholesterol
17.  Diversity is in My Veins: the Role of BMP Signaling during Venous Morphogenesis in Zebrafish Illustrates the Heterogeneity within Endothelial Cells 
Endothelial cells are a highly diverse group of cells which display distinct cellular responses to exogenous stimuli. While the aptly named Vascular Endothelial Growth Factor -A(VEGF-A) signaling pathway is hailed as the most important signaling input for endothelial cells, additional factors also participate in regulating diverse aspects of endothelial behaviors and functions. Given this heterogeneity, these additional factors appear to play a critical role in creating a custom-tailored environment to regulate behaviors and functions of distinct subgroups of endothelial cells. For instance, molecular cues that modulate morphogenesis of arterial vascular beds can be quite distinct from those that govern morphogenesis of venous vascular beds. Recently, we have found that Bone Morphogenetic Protein (BMP) signaling selectively promotes angiogenesis from venous vascular beds without eliciting similar responses from arterial vascular beds in zebrafish, indicating that BMP signaling functions as a context-dependent regulator during vascular morphogenesis. In this review, we will provide an overview of the molecular mechanisms that underlie pro-angiogenic effects of BMP signaling on venous vascular beds in the context of endothelial heterogeneity, and suggest a more comprehensive picture of the molecular mechanisms of vascular morphogenesis during development.
PMCID: PMC4140958  PMID: 25060789
Venous endothelial cells; endothelial heterogeneity; vascular morphogenesis; BMP signaling; zebrafish
18.  Links between ectopic fat and vascular disease in humans 
The average of overweight individual can have differential fat depots in target-organs or specific compartments of the body. This ectopic fat distribution may be more of a predictive factor for cardiovascular risk than obesity. Abdominal visceral obesity, a representative ectopic fat, is robustly associated with insulin resistance and cardiovascular risk. Fat depots in the liver and muscle tissue cause adverse cardiometabolic risk by affecting glucose and lipid metabolism. Pericardial fat and perivascular fat affect coronary atherosclerosis, cardiac function, and hemodynamics. Fat around the neck is associated with systemic vascular resistance. Fat around the kidney may increase blood pressure and induce albuminuria. Fat accumulation in or around the pancreas alters glucose metabolism, conferring cardiovascular risk.
Ectopic fat may act as an active endocrine and paracrine organ that releases various bioactive mediators that influence insulin resistance, glucose and lipid metabolism, coagulation, and inflammation, which all contribute to cardiovascular risk. As both obese and apparently lean individuals can have ectopic fat, regional fat distribution may play an important role in the development of cardiovascular diseases in both non-obese and obese people.
PMCID: PMC4140970  PMID: 25035342
ectopic fat; cardiovascular risk; mechanism; imaging
19.  A Brief Etymology of the Collateral Circulation 
It is well known that the protective capacity of the collateral circulation falls short in many individuals with ischemic disease of the heart, brain and lower extremities. In the past fifteen years, opportunities created by molecular and genetic tools, together with disappointing outcomes in many “angiogenic” trials, has led to a significant increase in the number of studies that focus on: 1) understanding the basic biology of the collateral circulation; 2) identifying the mechanisms that limit the collateral circulation’s capacity in many individuals; 3) devising methods to measure collateral extent, which has been found to vary widely among individuals; and 4) developing treatments to increase collateral blood flow in obstructive disease. Unfortunately, accompanying this increase in reports has been a proliferation of vague terms used to describe the disposition and behavior of this unique circulation, as well as the increasing miss-use of well-ensconced ones by new (and old) students of the collateral circulation. With this in mind, we provide a brief glossary of readily understandable terms to denote the formation, adaptive growth, and mal-adaptive rarefaction of the collateral circulation. We also propose terminology for several newly discovered processes that occur in the collateral circulation. Finally, we include terms used to describe vessels that are sometimes confused with collaterals, as well as terms describing processes active in the general arterial-venous circulation when ischemic conditions engage the collateral circulation. We hope this brief review will help unify the terminology used in collateral research.
PMCID: PMC4140974  PMID: 25012127
collateral circulation; ischemic heart disease; ischemic stroke; peripheral artery disease; angiogenesis
20.  Alternative splicing of endothelial fibronectin is induced by disturbed hemodynamics and protects against hemorrhage of the vessel wall 
Abnormally low flow conditions, sensed by the arterial endothelium, promote aneurysm rupture. Fibronectin (FN) is among the most abundant extracellular matrix proteins and is strongly upregulated in human aneurysms, suggesting a possible role in disease progression. Altered FN splicing can result in the inclusion of EIIIA and EIIIB exons, generally not expressed in adult tissues. We sought to explore the regulation of FN and its splicing and their possible roles in the vascular response to disturbed flow.
Approach and Results
We induced low and reversing flow in mice by partial carotid ligation, and assayed FN splicing in an endothelium-enriched intimal preparation. Inclusion of EIIIA and EIIIB was increased as early as 48hrs, with negligible increases in total FN expression. To test the function of EIIIA and EIIIB inclusion, we induced disturbed flow in EIIIAB-/- mice unable to include these exons and found that they developed focal lesions with hemorrhage and hypertrophy of the vessel wall. Acute deletion of floxed FN caused similar defects in response to disturbed flow, consistent with a requirement for the upregulation of the spliced isoforms, rather than a developmental defect. Recruited macrophages promote FN splicing, since their depletion by clodronate liposomes blocked the increase in endothelial EIIIA and EIIIB inclusion in the carotid model.
These results uncover a protective mechanism in the inflamed intima that develops under disturbed flow, by showing that splicing of FN mRNA in the endothelium, induced by macrophages, inhibits hemorrhage of the vessel wall.
PMCID: PMC4140979  PMID: 24903094
Fibronectin; Hemorrhage; Shear Stress; Macrophage; Alternative Splicing
21.  Regulation of HDL on hematopoietic stem/progenitor cells in atherosclerosis requires SR-BI expression 
Recently we demonstrated that scavenger receptor type BI (SR-BI), a HDL receptor, was expressed on murine hematopoietic stem/progenitor cells (HSPC) and infusion of reconstituted HDL and purified human apoA-I suppressed HSPC proliferation. We hypothesized that SR-B1 expression is required for the observed anti-proliferative effects of HDL on HSPC.
Approach and Results
SR-BI deficient (SR-BI−/−) mice and wild type (WT) controls were fed on chow or HFD (HFD) for 8–10 weeks. Under chow diet, a significant increase in Lin-Sca1+cKit+ cells (LSK cells, so called HSPC) was found in the BM of SR-BI−/− mice compared with WT mice. HFD induced a further expansion of CD150+CD48− LSK cells (HSCs), HSPCs, and granulocyte monocyte progenitors (GMPs) in SR-BI−/− mice. Injection of reactive oxygen species (ROS) inhibitor N-acetylcysteine attenuated HFD-induced HSPC expansion, leukocytosis and atherosclerosis in SR-BI−/− mice. ApoA-I infusion inhibited HSPC cell proliferation, Akt phosphorylation and ROS production in HSPC and plaque progression in low density lipoprotein receptor knockout (LDLr−/−) apoA-I−/− mice on HFD but had no effect on SR-BI−/− mice on HFD. Transplantation of SR-BI−/− BM cells into irradiated LDLr−/− recipients resulted in enhanced white blood cells (WBC) reconstitution, inflammatory cell production and plaque development. In patients with coronary heart disease, HDL levels were negatively correlated with WBC count and HSPC frequency in the peripheral blood. By flow cytometry, SR-BI expression was detected on human HSPC.
SR-BI plays a critical role in the HDL-mediated regulation HSPC proliferation and differentiation which is associated with atherosclerosis progression.
PMCID: PMC4140992  PMID: 24969774
HDL; hematopoietic stem/progenitor cells; atherosclerosis
22.  Association Between Shear Stress and Platelet-derived TGF-β1 Release and Activation in Animal Models of Aortic Valve Stenosis 
Aortic valve stenosis (AS) is characterized by fibrosis and calcification of valves leading to aortic valve (AV) narrowing, resulting in high wall shear stress (WSS) across the valves. We previously demonstrated that high shear stress can activate platelet-derived transforming growth factor-β1 (TGF-β1), a cytokine implicated inducing fibrosis and calcification. The aim of this study was to invest the role of shear-induced platelet release of TGF-β1 and its activation in AS.
Approach and Results
We studied hypercholesterolemic Ldlr−/−Apob100/100/Mttpfl/fl/Mx1Cre+/+ (Reversa) mice that develop AS on western diet (WD) and a surgical ascending aortic constriction (AAC) mouse model that acutely simulates the hemodynamics of AS to study shear-induced platelet TGF-β1 release and activation. Reversa mice on WD for 6 months had thickening of the AVs, increased WSS and increased plasma TGF-β1 levels. There were weak and moderate correlations between WSS and TGF-β1 levels in the progression and reversed Reversa groups, and a stronger correlation in the AAC model in WT mice, but not in mice with a targeted deletion of megakaryocyte and platelet TGF-β1 (Tgfb1flox). Plasma total TGF-β1 levels correlated with collagen deposition in the stenotic valves in Reversa mice. Although active TGF-β1 levels were too low to be measured directly, we found: 1. canonical TGF-β1 (p-Smad2/3) signaling in the leukocytes and canonical and non-canonical (p-Erk1/2) TGF-β1 signaling in AVs of Reversa mice on a WD, and 2. TGF-β1 signaling of both pathways in the AAC stenotic area in WT, but not Tgfb1flox mice.
Shear-induced, platelet-derived TGF-β1 activation may contribute to AS.
PMCID: PMC4141006  PMID: 24903096
aortic valve stenosis; platelet; transforming growth factor-β1; wall shear stress
23.  Receptor Tyrosine Kinase endocytosis in endothelium: biology and signaling 
Receptor tyrosine kinases (RTKs) are involved in regulation of key process in roles in endothelial biology including proliferation, migration and angiogenesis. It is now generally accepted that RTK signaling occurs intracellularly as well as on the plasma membrane although many important details remain to be worked out. Endocytosis and subsequent intracellular trafficking spatiotemporally regulate RTK signaling while “signaling endosomes” provide a platform for the compartmentalization of signaling events. This review summarizes recent advances in our understanding of endothelial RTK endocytosis and signaling using vascular endothelial growth factor receptor-2 as a paradigm.
PMCID: PMC4141018  PMID: 24925972
endocytosis; endosomes; signaling; VEGFR2; NRP1; synectin; Myosin-VI; FGFR1; EGFR
24.  The impact of phospholipid transfer protein on nascent HDL formation and remodeling 
Phospholipid transfer protein (PLTP), which binds phospholipids and facilitates their transfer between lipoproteins in plasma, plays a key role in lipoprotein remodeling but its influence on nascent HDL formation is not known. The effect of PLTP over-expression on apoA-I lipidation by primary mouse hepatocytes was investigated.
Approach and Results
Over-expression of PLTP through an adenoviral vector markedly affected the amount and size of lipidated apoA-I species that were produced in hepatocytes in a dose-dependent manner, ultimately generating particles that were smaller than 7.1 nm but larger than lipid-free apoA-I. These < 7.1 nm small particles generated in the presence of over-expressed PLTP were incorporated into mature HDL particles more rapidly than apoA-I both in vivo and in vitro, and were less rapidly cleared from mouse plasma than lipid-free apoA-I. The < 7.1 nm particles promoted both cellular cholesterol and phospholipid efflux in an ATP-binding cassette transporter A1 (ABCA1) dependent manner, similar to apoA-I in the presence of PLTP. Lipid-free apoA-I had a greater efflux capacity in the presence of PLTP than in the absence of PLTP, suggesting that PLTP may promote ABCA1-mediated cholesterol and phospholipid efflux. These results indicate that PLTP alters nascent HDL formation by modulating the lipidated species as well as promoting the initial process of apoA-I lipidation.
Our findings suggest that PLTP exerts significant effects on apoA-I lipidation and nascent HDL biogenesis in hepatocytes by promoting ABCA1-mediated lipid efflux and the remodeling of nascent HDL particles.
PMCID: PMC4141034  PMID: 25060793
PLTP; HDL formation; cholesterol efflux; hepatocyte
25.  TMEM55B is a Novel Regulator of Cellular Cholesterol Metabolism 
Inter-individual variation in pathways impacting cellular cholesterol metabolism can influence levels of plasma cholesterol, a well-established risk factor for cardiovascular disease. Inherent variation among immortalized lymphoblastoid cell lines (LCLs) from different donors can be leveraged to discover novel genes that modulate cellular cholesterol metabolism. The objective of this study was to identify novel genes that regulate cholesterol metabolism by testing for evidence of correlated gene expression with cellular levels of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) mRNA, a marker for cellular cholesterol homeostasis, in a large panel of LCLs.
Approach and Results
Expression array profiling was performed on 480 LCLs established from participants of the Cholesterol and Pharmacogenetics statin clinical trial, and transcripts were tested for evidence of correlated expression with HMGCR as a marker of intracellular cholesterol homeostasis. Of these, transmembrane protein 55b (TMEM55B) showed the strongest correlation (r=0.29, p=4.0E-08) of all genes not previously implicated in cholesterol metabolism and was found to be sterol regulated. TMEM55B knock-down in human hepatoma cell lines promoted the decay rate of the low density lipoprotein receptor (LDLR), reduced cell surface LDLR protein, impaired LDL uptake, and reduced intracellular cholesterol.
Here we report identification of TMEM55B as a novel regulator of cellular cholesterol metabolism through the combination of gene expression profiling and functional studies. The findings highlight the value of an integrated genomic approach for identifying genes that influence cholesterol homeostasis.
PMCID: PMC4141484  PMID: 25035345
cholesterol; LDLR; post-transcriptional regulation; cardiovascular disease; PIP2

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