Human Vascular Tissue Research Articles

Pulsatile Atheroprone Shear Stress Affects the Expression of Transient Receptor Potential Channels in Human Endothelial Cells

The goal of the study was to assess whether pulsatile atheroprone shear stress modulates the expression of transient receptor potential (TRP) channels, TRPC3, TRPC6, TRPM7, and TRPV1 mRNA, in human umbilical vascular endothelial cells. Exposure of cultured vascular endothelial cells to defined shear stress, producing a constant laminar flow (generating a shear stress of 6 dyne/cm(2)), laminar pulsatile atheroprotective flow (with a mean shear stress of 20 dyne/cm(2)), or laminar atheroprone bidirectional flow (with a mean shear stress of 0 dyne/cm(2)) differentially induced TRPC6 and TRPV1 mRNA as measured by quantitative real-time RT-PCR and normalized to GAPDH expression. Thereby, TRPC6 and TRPV1 mRNA expressions were significantly increased after 24 hours of exposure to an atheroprone flow profile compared with an atheroprotective flow profile. Furthermore, the expression of transcription factors GATA1 and GATA4 was significantly correlated with the expression of TRPC6 mRNA. In contrast, after 24 hours of constant laminar flow, the expression of TRPC6 and TRPV1 mRNA was unchanged, whereas the expression of TRPC3 and TRPM7 was significantly higher in endothelial cells exposed to shear stress in comparison with endothelial cells grown under static conditions. There was a significant association between the expression of TRPC6 and tumor necrosis factor-α mRNA in human vascular tissue. No-flow and atheroprone flow conditions are equally characterized by an increase in the expression of tumor necrosis factor-α; however, inflammation-associated endothelial cell reactions may be further aggravated at atheroprone flow conditions by the increase of TRPV1 and TRPC6, as observed in our study.

Circulation : Clinical Summaries

<i>Circulation</i> : Clinical Summaries

Influence of matrix metalloproteinase-12 on fibrinogen level

In vitro studies have shown that matrix metalloproteinase-12 (MMP12) can degrade fibrinogen, a clotting factor whose level predicts risk of advanced atherosclerosis and myocardial infarction. In this study, we found that mean plasma fibrinogen level was approximately 10-fold higher in MMP12 knockout mice than wildtype mice ( p = 0.0006). Differential allelic expression analysis of human MMP12 gene polymorphism rs17368582 in human vascular tissues showed an allele-specific effect on MMP12 expression, with one allele (T) having 1.6 fold higher expression level than the other allele (C) ( p = 0.0006). In a population cohort, we found that individuals homozygous for the MMP12 low expression allele had higher plasma fibrinogen levels (2.95 mg/mL compared with 2.61 mg/mL in other individuals, p = 0.029) and increased risk of advanced atherosclerosis [odds ratio 6.3 (95% CI 1.9–20.8), p = 0.003] and myocardial infarction [hazard ratio 5.6 (95% CI 1.7–18.3), p = 0.005]. In summary, our study in mouse and humans provides in vivo evidence of an effect of MMP12 on fibrinogen level.

Decreased Expression of Transient Receptor Potential Channels in Cerebral Vascular Tissue from Patients After Hypertensive Intracerebral Hemorrhage

Recent data indicate that transient receptor potential (TRP) cation channels play an important role in hypertension. Now, we tested the hypothesis that TRP expression is altered in human cerebral vascular tissue in patients who had experienced hypertensive intracerebral hemorrhage. TRPC1, TRPC3, TRPC5, TRPC6, TRPM4, TRPM6, and TRPM7 channels were detected in cerebral vascular tissue by quantitative real-time RT-PCR. Control cerebral vascular tissue was obtained from normotensive patients who underwent neurosurgical operation because of brain tumor. To examine a possible relation between the expression of TRP expression and hypoxic conditions caused by the intracerebral bleeding, we examined the expression of hypoxia inducible factor 1a (HIF1a). Transcripts of TRPC3, TRPC5, TRPM6, and HIF1a were significantly reduced in cerebral vascular tissue from patients after hypertensive intracerebral hemorrhage compared to controls. TRPC3 mRNA correlated well with the expression of HIF1a mRNA (r2 = 0.59; p = 0.01). TRPC3 expression is associated with hypertension and hypoxic conditions in human cerebral vascular tissue.

Expression of FGF23/KLOTHO system in human vascular tissue

BackgroundFibroblast growth factor (FGF)-23 levels have been associated with impaired vasoreactivity, increased arterial stiffness, and cardiovascular morbi-mortality, whereas a protective function of KLOTHO against endothelial dysfunction has been reported. Since expression of the FGF23–KLOTHO system in human vascular tissue remains unproved, we aimed to study the expression of FGF23, FGF receptors (FGFR) and KLOTHO in human aorta. In addition, we analyzed the FGF23–KLOTHO expression in occlusive coronary thrombi. MethodsThoracic aorta specimens from 44 patients underwent elective cardiac surgery, and thrombus material from 2 patients with acute coronary syndrome (ACS), were tested for FGF23–KLOTHO system expression. ResultsExpression of KLOTHO (mean expression level 4.85±5.43, arbitrary units) and two of the three cognate FGFR (FGFR-1 and -3) were detected and confirmed by RT-PCR, sequencing and qRT-PCR. KLOTHO expression was confirmed within occlusive coronary thrombi from patients with ACS. However, expression of FGF23 and FGFR4 was not observed. We also detected the aortic expression of membrane-anchored A Desintegrin and Metalloproteinases (ADAM)-17, the enzyme responsible for the shedding of KLOTHO from the cell surface, and the anti-inflammatory cytokine interleukin (IL)-10. Interestingly, in aortic samples there was a direct association between KLOTHO mRNA levels and those of ADAM-17 and IL-10 (r=0.54, P<0.001; r=0.51, P<0.01, respectively). ConclusionsHuman vascular tissue expresses members of the FGF23–KLOTHO system, indicating that it can be a direct target organ for FGF23. In addition, KLOTHO expression is also detected in occlusive coronary thrombi. These findings suggest a putative role of FGF23–KLOTHO axis in human vascular pathophysiology and cardiovascular disease.

Identification of mitogen-activated protein/extracellular signal-responsive kinase kinase 2 as a novel partner of the scaffolding protein human homolog of disc-large

Human disc-large homolog (hDlg), also known as synapse-associated protein 97, is a scaffold protein, a member of the membrane-associated guanylate kinase family, implicated in neuronal synapses and epithelial-epithelial cell junctions whose expression and function remains poorly characterized in most tissues, particularly in the vasculature. In human vascular tissues, hDlg is highly expressed in smooth muscle cells (VSMCs). Using the yeast two-hybrid system to screen a human aorta cDNA library, we identified mitogen-activated protein/extracellular signal-responsive kinase (ERK) kinase (MEK)2, a member of the ERK cascade, as an hDlg binding partner. Site-directed mutagenesis showed a major involvement of the PSD-95, disc-large, ZO-1 domain-2 of hDlg and the C-terminal sequence RTAV of MEK2 in this interaction. Coimmunoprecipitation assays in both human VSMCs and human embryonic kidney 293 cells, demonstrated that endogenous hDlg physically interacts with MEK2 but not with MEK1. Confocal microscopy suggested a colocalization of the two proteins at the inner layer of the plasma membrane of confluent human embryonic kidney 293 cells, and in a perinuclear area in human VSMCs. Additionally, hDlg also associates with the endoplasmic reticulum and microtubules in these latter cells. Taken together, these findings allow us to hypothesize that hDlg acts as a MEK2-specific scaffold protein for the ERK signaling pathway, and may improve our understanding of how scaffold proteins, such as hDlg, differentially tune MEK1/MEK2 signaling and cell responses.

Three-Dimensional Topography of Synthetic Scaffolds Induces Elastin Synthesis by Human Coronary Artery Smooth Muscle Cells

Due to the important structural and signaling roles of elastin in vascular stability, engineered human vascular tissues must incorporate elastin. However, despite considerable progress toward engineering of elastin-containing vascular tissues from animal cells, currently engineered vascular tissues using human cells largely lack elastin. In this study, we evaluated the effect of scaffold topography (two dimensional [2D] vs. three dimensional [3D]) on elastogenesis in adult human coronary artery smooth muscle cells (HCASMCs). We report that elastin gene expression by HCASMCs was increased by twofold after 4 days of culture in porous 3D polyurethane scaffolds. Transforming growth factor β1 (TGF-β1) further increased elastin gene expression in 3D cultures but not in 2D cultures. To evaluate if gene expression is translated into elastin synthesis, both 2D and 3D cultures were analyzed using Western blots. We show that only HCASMCs in 3D scaffolds produced elastin, suggesting that scaffold geometry itself is an important cue for elastogenesis. Moreover, TGF-β1 enhanced elastin synthesis in 3D, but had no effect on cells grown on 2D surfaces. TGF-β1, known to induce vascular smooth muscle cells (VSMC) differentiation, upregulated contractile VSMC marker proteins smooth muscle-α-actin and calponin in cells on 2D surfaces. Interestingly, in 3D scaffolds, TGF-β1 failed to upregulate these differentiation marker proteins for at least 7 days, but did so in cells cultured for 14 days, whereas elastin synthesis was not affected. To our knowledge this study is the first to successfully demonstrate that adult human VSMC can produce elastin when seeded on 3D scaffolds and to directly compare the effect of scaffold topography on elastin synthesis. Knowledge about the conditions required to regulate the phenotype of human VSMCs is paramount to engineer elastin-containing autologous human vascular substitutes.

Modulation of the apelin/APJ system in heart failure and atherosclerosis in man

The aim of this study was to determine whether the apelin/APJ system is altered in human cardiovascular disease by investigating whether the expression of apelin or its receptor is altered at the protein level. Radioligand binding studies were used to determine apelin receptor density in human cardiac tissues. Apelin peptide levels in cardiovascular tissues were determined by radioimmunoassay. In vitro pharmacology was used to assess vasoactive properties of apelin in human coronary artery. Localization of apelin and its receptor in coronary artery was determined using immunohistochemistry. Apelin receptor density was significantly decreased in left ventricle from patients with dilated cardiomyopathy or ischaemic heart disease compared with controls, but apelin peptide levels remained unchanged. Apelin was up-regulated in human atherosclerotic coronary artery and this additional peptide localized to the plaque, colocalizing with markers for macrophages and smooth muscle cells. Apelin potently constricted human coronary artery. We have detected changes in the apelin/APJ system in human diseased cardiac and vascular tissue. The decrease in receptor density in heart failure may limit the positive inotropic actions of apelin, contributing to contractile dysfunction. The contribution of the increased apelin levels in atherosclerotic coronary artery to disease progression remains to be determined. These data suggest a potential role for the apelin/APJ system in human cardiovascular disease.

Dihydrotestosterone alters cyclooxygenase-2 levels in human coronary artery smooth muscle cells

Both protective and nonprotective effects of androgens on the cardiovascular system have been reported. Our previous studies show that the potent androgen receptor (AR) agonist dihydrotestosterone (DHT) increases levels of the vascular inflammatory mediator cyclooxygenase (COX)-2 in rodent cerebral arteries independent of an inflammatory stimulus. Little is known about the effects of androgens on inflammation in human vascular tissues. Therefore, we tested the hypothesis that DHT alters COX-2 levels in the absence and presence of induced inflammation in primary human coronary artery smooth muscle cells (HCASMC). Furthermore, we tested the ancillary hypothesis that DHT's effects on COX-2 levels are AR-dependent. Cells were treated with DHT (10 nM) or vehicle for 6 h in the presence or absence of LPS or IL-1beta. Similar to previous observations in rodent arteries, in HCASMC, DHT alone increased COX-2 levels compared with vehicle. This effect of DHT was attenuated in the presence of the AR antagonist bicalutamide. Conversely, in the presence of LPS or IL-1beta, increases in COX-2 were attenuated by cotreatment with DHT. Bicalutamide did not affect this response, suggesting that DHT-induced decreases in COX-2 levels occur independent of AR stimulation. Thus we conclude that DHT differentially influences COX-2 levels under physiological and pathophysiological conditions in HCASMC. This effect of DHT on COX-2 involves AR-dependent and- independent mechanisms, depending on the physiological state of the cell.

The flavonoid dioclein is a selective inhibitor of cyclic nucleotide phosphodiesterase type 1 (PDE1) and a cGMP-dependent protein kinase (PKG) vasorelaxant in human vascular tissue

The inhibitory effect of the flavonoid dioclein was assessed on purified vascular cyclic nucleotide phosphodiesterase isoforms (EC 3.1.4.17, PDE1-5) in comparison with 8-methoxymethyl-isobutylmethylxanthine (8-MM-IBMX) and vinpocetine which are currently used as PDE1 inhibitors. The mechanism underlying the vasorelaxant effect of dioclein was investigated in human saphenous vein. Dioclein inhibited PDE1 more selectively than vinpocetine and 8-MM-IBMX, with IC 50 values of 2.47 ± 0.26 and 1.44 ± 0.35 µM, respectively in basal- and calmodulin-activated states. Dioclein behaved as a competitive inhibitor for cGMP hydrolysis by PDE1 in basal- and calmodulin-activated states ( K i = 0.62 ± 0.14 and 0.55 ± 0.07 µM, respectively), indicating this inhibitory effect to be independent of calmodulin interactions. In addition, dioclein induced a concentration-dependent relaxation of human saphenous vein which was independent on the presence of functional endothelium (EC 50 values of 7.3 ± 3.1 and 11 ± 2.7 µM, respectively with and without endothelium). 8-MM-IBMX relaxed human saphenous vein with an EC 50 = 31 ± 16 µM, whereas vinpocetine did not cause any vasorelaxation at concentrations up to 100 µM. Rp-8-pCPT-cGMPS, which inhibits cGMP-dependent protein kinase (PKG), blocked the vasodilator effect of dioclein, whereas H-89, which is a cAMP-dependent protein kinase (PKA) inhibitor, had a minor inhibitory effect. Our data show that dioclein is a potent calmodulin-independent selective inhibitor of PDE1 and that inhibition of PDE1 is involved in the PKG-mediated vasorelaxant effect of dioclein in human saphenous vein. Furthermore, dioclein may represent a new archetype to develop more specific PDE1 inhibitors.

Renal and vascular glutathione S-transferase μ is not affected by pharmacological intervention to reduce systolic blood pressure

Our previous studies demonstrated reduced rat glutathione S-transferase mu type 1 (Gstm1) expression in stroke-prone spontaneously hypertensive rats (SHRSPs), when compared with the normotensive Wistar-Kyoto rat. This study investigated the effects of angiotensin II type 1 receptor blocker (ARB) and a diuretic/vasodilator combination on the expression levels of rat Gstm1 and other Gstm isoforms. Antihypertensive treatments of young and mature SHRSPs with an ARB and a diuretic/vasodilator combination improved SBP but did not affect the expression levels of Gstm1. Although Gstm1 is a member of a family of highly homologous genes, with the exception of Gstm2, there was no evidence for compensatory increase in expression of other Gstm isoforms. In contrast, we observed reduced expression of several other Gstm isoforms in untreated SHRSPs. Untreated SHRSPs demonstrated increased renal and vascular oxidative stress, both of which were not significantly affected by the antihypertensive treatments. Untreated SHRSPs scored significantly higher when assessed for renal histopathological damage, and this was improved by antihypertensive treatments. These results suggest that reduced Gstm1 expression in SHRSPs is due to strain-dependent genetic abnormalities, playing a causative role in the development of hypertension, probably through oxidative stress pathway. Renal changes occur as a consequence of increased blood pressure and can be improved when treated with antihypertensive drugs. In silico comparative genome analysis combined with expression studies in rat and human vascular tissue revealed that there are possible four human homologues (GSTM1, GSTM2, GSTM4 and GSTM5) for rat Gstm1.

Expression and stability of two isoforms of ABCG1 in human vascular cells

Objective To evaluate the expression of two ABCG1 isoforms that differ in the presence or absence of a 12 amino acid (AA) peptide between the ABC cassette and the transmembrane region, termed ABCG1(+12) and ABCG1(−12), respectively, in human vascular cells and tissues. Methods and results mRNA for both isoforms was expressed in human macrophages, vascular endothelial and smooth muscle cells as well as whole human spleen, lung, liver and brain tissue. However, ABCG1(+12) was not expressed in mouse tissues. 2D gel electrophoresis of ABCG1 protein indicated that both protein isoforms were expressed in human macrophages. Furthermore the half-lives of the two ABCG1 protein isoforms, stably expressed in CHOK1 cells, measured under basal conditions were different, suggesting the presence of a degradation or stabilising signal in or near the 12AA region of ABCG1(+12). Conclusion ABCG1(+12) is an isoform of ABCG1 exclusively expressed in human cells at the RNA and protein level. As ABCG1(+12) is not expressed in mice, although mouse models are widely used to elucidate the function of ABCG1, further investigations into the importance of this human ABCG1 isoform are warranted.

Use of Human Vascular Tissue Microarrays for Measurement of Advanced Glycation Endproducts

Advanced glycation endproducts (AGEs) are present in the vasculature and are associated with vascular disease. We determined levels of AGEs in eight distinct adult vascular tissues using tissue microarray (TMA) technology and associated these levels with clinical characteristics. Medium-to-large caliber blood vessels were harvested from 100 adult autopsies to create 17 TMAs. AGE levels were evaluated by IHC using a polyclonal anti-AGE antibody on over 700 unique blood vessels. Slides were digitally scanned, and quantitative analysis was performed using a color deconvolution image analysis technique. Medial AGE staining was strongly correlated between all eight blood vessels. In the media, AGE staining levels were significantly higher at older ages (p=0.009), in white subjects (p<0.001) and with longer postmortem interval (PMI; p<0.0001). These associations remained significant after simultaneous adjustment for age, race/ethnicity, PMI, and diabetes status. Diabetes was associated with elevated AGE levels but only after adjustment for confounding by clinical variables including race/ethnicity, hypertension, and kidney function. This extensive vascular study shows that AGE accumulation in the macrovasculature is a global process affecting atherosclerosis-prone and -resistant vessels. It also suggests ethnicity has a previously undescribed role in vascular tissue AGE levels. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

The effect of short-term, high glucose concentration on endothelial cells and leukocytes in a 3D in vitro human vascular tissue model

Efforts to determine a link between diabetes and atherosclerosis have involved examining the effect of high glucose levels on the adhesion and migration of circulating leukocytes, mostly monocytes and T lymphocytes. Leukocyte differentiation and proliferation within the subendothelial space can also be investigated by the use of a 3D in vitro human vascular tissue model. This model was used to study the effect of short-term, high glucose concentration on certain cell behavior associated with the early stages of atherosclerosis. Samples were exposed to either a 30- or 5.6-mM glucose concentration for 9 h to represent either hyperglycemic or normoglycemic conditions, respectively. There was a significant increase in vascular cell adhesion molecule-1 expression on the endothelial cells exposed to a 30-mM compared to a 5.6-mM glucose concentration. There was no significant difference in either intercellular adhesion molecule-1 or E-selectin expression on the endothelial cells exposed to a 30-mM compared to a 5.6-mM glucose concentration. After the endothelium was exposed to 30 mM glucose concentration, there was a 70% increase in the number of monocytes (CD14(+)) migrating across the endothelium and a 28% increase in the number of these monocytes differentiating into macrophages, compared to cell migration and differentiation across the endothelium exposed to 5.6 mM glucose concentration. Also, for the endothelium exposed to 30 mM glucose concentration, there were nearly 2.5 times more T lymphocytes that migrated across the endothelium, along with significant cell proliferation, compared to cell migration across the endothelium exposed to 5.6 mM glucose concentration.

Increased dynamic stiffness of extracardial vessels in left main stem coronary artery disease

Objective: Dynamic stiffness (DS) and the elastic properties of extracardial vessels in patients with coronary artery disease with or without left main stenosis were analyzed by direct mechanical measurements of human vascular tissue.

Interaction of bacteria of the genus Bartonella with host cells: Inhibition of apoptosis, induction of proliferation, and oncogenesis

Bacteria of the genus Bartonella are responsible for many previously undetected or recurrent human diseases whose harmfulness increases due to an increasing number of immunocompromised persons. As intracellular parasites, Bartonella species colonize endothelial cells of human and animal vascular tissues and erythrocytes.

Human saphenous vein relaxation in response to sodium nitroprusside correlates with systolic blood pressure and phosphorylated heat shock protein 27

Stress has been shown to cause hypertension and increase heat shock protein 27 (HSP27) expression and phosphorylation in animal models. Impaired relaxation of vascular smooth muscle occurs during hypertension and may be associated with HSP27 expression and/or phosphorylation. Human saphenous vein (HSV) was used as a model system to examine impaired relaxation and HSP27 expression and phosphorylation in a hypertensive patient population. Relaxation of HSV samples was determined by measurements of tension produced in a muscle bath in response to submaximal norepinephrine dose followed by a sodium nitroprusside dose curve. Relaxation was found to be significantly inversely correlated with systolic blood pressure at patient admission (R2=0.6175, n=10, p&lt;0.05). Western blotting for HSP27 and phosphorylated HSP27 revealed a significant inverse correlation between relaxation and the amount of phosphoryated HSP27 present in the HSV samples (R2=0.8699, n=5, p&lt;0.05). Hence this study provides a link between hypertension, HSP27 phosphorylation, and impaired relaxation in human vascular tissue. The mechanism for this impaired relaxation may involve the expression and/or phosphorylation of HSP27 since phosphoHSP27 also appears to be associated with impaired relaxation. Support: NIH RO1 70715

A new mRNA splice variant coding for the human EP 3-I receptor isoform

The cellular localization of prostaglandin E 2 receptors (EP) and their corresponding transcripts were investigated in human gastric and vascular tissues. A strong staining of the EP 3 receptor on the gastric glands, mucous cells, media of the mammary and pulmonary arteries was observed by immunohistochemistry. We identified a new mRNA splice variant of the EP 3 gene in human gastric fundic mucosa, mammary artery and pulmonary vessels. This EP 3-Ic transcript contains exons 1, 2, 3, 5 and 6 of the EP 3 gene and should be translated in the EP 3-I isoform. In addition, the EP 3-Ib, EP 3-II, EP 3-III, EP 3-IV and EP 3-e mRNAs were detected in these tissues.

Non‐genomic effects of androgens on isolated human vascular and nonvascular penile erectile tissue

To evaluate non-genomic effects of testosterone and dihydrotestosterone (DHT) on isolated human cavernosal arteries (HCA) and corpus cavernosum (HCC) using organ-bath studies and radio-immunoassays (RIA), as non-genomic effects of androgens are reported for vascular smooth musculature and there is evidence that the relaxant response involves a modulation of cyclic nucleotide tissue levels. The relaxation induced by the cumulative addition of testosterone and DHT (0.01-10 microm) was studied using circular segments of HCA and strip preparations of HCC. To evaluate the effects of testosterone and DHT on tissue levels of cAMP and cGMP, specimens were exposed to increasing concentrations of the hormones. Forskolin and sodium nitroprusside (SNP) served as reference compounds. Testosterone and DHT dose-dependently reversed the noradrenaline-induced tension of vascular segments and HCC strips. At the maximum concentration, testosterone and DHT reduced the mean (sd) tension to 79.8 (4.43)% and 83.9 (10.94)%, respectively. SNP and forskolin significantly stimulated the production of cGMP and cAMP. No effects of testosterone and DHT on cGMP and cAMP levels were detected. Rapid androgen-induced relaxation of HCA and HCC occurs via non-genomic mechanisms. In penile erectile tissue, non-genomic relaxant effects of testosterone and DHT are not mediated via modulation of cyclic nucleotide tissue levels. Additional studies are required to establish if non-genomic relaxant effects are important in ensuring a basal level of perfusion to maintain overall penile function.

44 KRUPPEL-LIKE FACTOR 4 REGULATES ENDOTHELIAL INFLAMMATION.

<h3>Background</h3> The vascular endothelium plays a critical role in vascular homeostasis. A limited proinflammatory, prothrombotic state is appropriate in the context of infection or wound healing, but sustained endothelial activation leads to deleterious conditions such as atherosclerosis and pathologic thrombosis. Inflammatory cytokines and nonlaminar blood flow induce endothelial dysfunction and confer a proadhesive and prothrombotic phenotype. Therefore, identification of factors that mediate the effects of these stimuli on endothelial function is of considerable interest. Kruppel-like factors (KLFs) are a subclass of the zinc-finger family of transcription factors. Previous studies demonstrate that KLF proteins typically regulate critical aspects of cellular differentiation and tissue development. Studies from our laboratory and others demonstrate an emerging role for this family of transcriptional regulators in vascular biology. KLF4/GKLF (gut-enriched Kruppel-like factor) was first identified as being highly expressed in epithelial cells, and subsequent work has verified a critical role in skin and intestinal development. KLF4 has also been identified in endothelial cells, yet its function in vessel biology has yet to be elucidated. <h3>Methods and Results</h3> Immunohistochemical analysis of mouse and human vascular tissues demonstrates the expression, in vivo, of KLF4 in human and mouse endothelial cells. Northern blot analysis of total mRNA harvested from primary endothelial cell lines derived from various human arterial vascular beds (aorta, pulmonary artery, and umbilical artery) shows expression of endothelial KLF4 in a variety of arterial and venous cell lines. Furthermore, we demonstrate that endothelial KLF4 is induced by proinflammatory stimuli and shear stress. Overexpression of KLF4 induces expression of multiple anti-inflammatory and antithrombotic factors, including eNOS and thrombomodulin, and inhibits basal and cytokine-mediated expression of a diverse set of proinflammatory factors, including MCP-1, RANTES, CRP, PAI-1, IL-6, and IL-8. The significance of endogenous expression of KLF4 on target genes was assessed in experiments using siRNA-mediated knockdown of KLF4. These experiments demonstrate that KLF4 depletion leads to enhancement of TNF-α-induced VCAM-1 and tissue factor expression. In addition to the determination of target genes, we have verified the functional importance of KLF4 by demonstrating that KLF4 expression markedly decreases inflammatory cell adhesion to the endothelial surface and prolongs clotting time under inflammatory states. As a first step toward understanding the molecular basis of KLF4-mediated regulation of endothelial target genes, we assessed the effect of KLF4 on target gene promoters. KLF4 differentially regulates the promoter activity of pro- and anti-inflammatory genes in a manner consistent with its anti-inflammatory function. <h3>Conclusion</h3> Inflammatory cytokines and the biomechanical effects of laminar shear stress are the most potent effectors of endothelial homeostasis identified to date. Perturbation of endothelial function by proinflammatory cytokines or nonlaminar flow has been documented in most vascular disease states, including vasculitis, aneurysm formation, pathologic thrombosis, and atherosclerosis. The observations described above implicate KLF4 as a novel regulator of endothelial activation in response to proinflammatory stimuli. A greater understanding of KLF4 function is thus of considerable scientific and potentially therapeutic interest.