Endothelial cells (ECs) respond to changes in mechanical forces, leading to the modulation of signaling networks and cell function; an example is the inhibition of EC proliferation by steady laminar flow. MicroRNAs (miRs) are short noncoding 20–22 nucleotide RNAs that negatively regulate the expression of target genes at the posttranscriptional level. This study demonstrates that miRs are involved in the flow regulation of gene expression in ECs. With the use of microRNA chip array, we found that laminar shear stress (12 dyn/cm ² , 12 h) regulated the EC expression of many miRs, including miR-19a. We further showed that stable transfection of miR-19a significantly decreased the expression of a reporter gene controlled by a conserved 3′-untranslated region of the cyclinD1 gene and also the protein level of cyclin D1, leading to an arrest of cell cycle at G1/S transition. Laminar flow suppressed cyclin D1 protein level, and this suppressive effect was diminished when the endogenous miR-19a was inhibited. In conclusion, we demonstrated that miR-19a plays an important role in the flow regulation of cyclin D1 expression. These results revealed a mechanism by which mechanical forces modulate endothelial gene expression.

Objective— Endothelial activation is implicated in atherogenesis and diabetes. The role of peroxisome proliferator-activated receptor-δ (PPAR-δ) in endothelial activation remains poorly understood. In this study, we investigated the anti-inflammatory effect of PPAR-δ and the mechanism involved. Methods and Results— In human umbilical vein endothelial cells (HUVECs), the synthetic PPAR-δ ligands GW0742 and GW501516 significantly inhibited tumor necrosis factor (TNF)-α–induced expression of vascular cell adhesion molecule-1 and E-selectin (assayed by real-time RT-PCR and Northern blotting), as well as the ensuing endothelial-leukocyte adhesion. Activation of PPAR-δ upregulated the expression of antioxidant genes superoxide dismutase 1, catalase, and thioredoxin and decreased reactive oxygen species production in ECs. Chromatin immunoprecipitation assays showed that GW0742 switched the association of BCL-6, a transcription repressor, from PPAR-δ to the vascular cell adhesion molecule (VCAM)-1 promoter. Small interfering RNA reduced endogenous PPAR-δ expression but potentiated the suppressive effect of GW0742 on EC activation, which suggests that the nonliganded PPAR-δ may have an opposite effect. Conclusions— We have demonstrated that ligand activation of PPAR-δ in ECs has a potent antiinflammatory effect, probably via a binary mechanism involving the induction of antioxidative genes and the release of nuclear corepressors. PPAR-δ agonists may have a potential for treating inflammatory diseases such as atherosclerosis and diabetes.

Objective—: Inflammatory responses play a pivotal role in the pathogenesis of hypertensive cardiac remodeling. Macrophage recruitment and polarization contribute to the development of cardiac fibrosis. Although serum-glucocorticoid regulated kinase 1 (SGK1) is a key mediator of fibrosis, its role in regulating macrophage function leading to cardiac fibrosis has not been investigated. We aimed to determine the mechanism by which SGK1 regulates the cardiac inflammatory process, thus contributing to hypertensive cardiac fibrosis. Methods and Results—: After angiotensin II infusion in mice, cardiac hypertrophy and fibrosis developed in wild-type but not SGK1 knockout mice, with equal levels of hypertension in both groups. Compared with wild-type hearts, SGK1 knockout hearts showed less infiltration of leukocytes and macrophages. Importantly, SGK1 deficiency led to decreased proportion of alternatively activated (M2) macrophages and increased levels of profibrotic cytokines. Angiotensin II infusion induced phosphorylation and nuclear localization of signal transducer and activator of transcription 3 (STAT3) whereas SGK1 knockout hearts showed this effect attenuated. In a 3-dimensional peptide gel culture, inhibition of STAT3 suppressed differentiation into M2 macrophages. Coculture of macrophages with cardiac fibroblasts in 3-dimensional peptide gel stimulated the expression of α-smooth muscle actin and collagen in cardiac fibroblasts. However, SGK1 knockout mice with macrophage deficiency showed reduced fibroblast-to-myofibroblast transition. Conclusion—: SGK1 may play an important role in macrophage recruitment and M2 macrophage activation by activating the STAT3 pathway, which leads to angiotensin II–induced cardiac fibrosis.

We sought to determine whether Dopamine D2 Receptor (D2R) agonists inhibit lung tumor progression and identify subpopulations of lung cancer patients that benefit most from D2R agonist therapy. We demonstrate D2R agonists abrogate lung tumor progression in syngeneic (LLC1) and human xenograft (A549) orthotopic murine models through inhibition of tumor angiogenesis and reduction of tumor infiltrating myeloid derived suppressor cells. Pathological examination of human lung cancer tissue revealed a positive correlation between endothelial D2R expression and tumor stage. Lung cancer patients with a smoking history exhibited greater levels of D2R in lung endothelium. Our results suggest D2R agonists may represent a promising individualized therapy for lung cancer patients with high levels of endothelial D2R expression and a smoking history.

Rationale : Phenotypic modulation of smooth muscle cells (SMCs), which are located in close proximity to endothelial cells (ECs), is critical in regulating vascular function. The role of flow-induced shear stress in the modulation of SMC phenotype has not been well defined. Objective : The objective was to elucidate the role of shear stress on ECs in modulating SMC phenotype and its underlying mechanism. Methods and Results : Application of shear stress (12 dyn/cm ² ) to ECs cocultured with SMCs modulated SMC phenotype from synthetic to contractile state, with upregulation of contractile markers, downregulation of proinflammatory genes, and decreased percentage of cells in the synthetic phase. Treating SMCs with media from sheared ECs induced peroxisome proliferator-activated receptor (PPAR)-α, -δ, and -γ ligand binding activities; transfecting SMCs with specific small interfering (si)RNAs of PPAR-α and -δ, but not -γ, inhibited shear induction of contractile markers. ECs exposed to shear stress released prostacyclin (PGI ₂ ). Transfecting ECs with PGI ₂ synthase-specific siRNA inhibited shear-induced activation of PPAR-α/δ, upregulation of contractile markers, downregulation of proinflammatory genes, and decrease in percentage of SMCs in synthetic phase. Mice with PPAR-α deficiency (compared with control littermates) showed altered SMC phenotype toward a synthetic state, with increased arterial contractility in response to angiotensin II. Conclusions : These results indicate that laminar shear stress induces synthetic-to-contractile phenotypic modulation in SMCs through the activation of PPAR-α/δ by the EC-released PGI ₂ . Our findings provide insights into the mechanisms underlying the EC-SMC interplays and the protective homeostatic function of laminar shear stress in modulating SMC phenotype.

The lymphatic vasculature is essential for maintaining interstitial fluid homeostasis, and dysfunctional lymphangiogenesis contributes to various pathological processes, including inflammatory disease and tumor metastasis. Mutations in FOXC2 are dominantly associated with late-onset lymphedema; however, the precise role of FOXC2 and a closely related factor, FOXC1, in the lymphatic system remains largely unknown. Here we identified a molecular cascade by which FOXC1 and FOXC2 regulate ERK signaling in lymphatic vessel growth. In mice, lymphatic endothelial cell–specific (LEC-specific) deletion of Foxc1, Foxc2, or both resulted in increased LEC proliferation, enlarged lymphatic vessels, and abnormal lymphatic vessel morphogenesis. Compared with LECs from control animals, LECs from mice lacking both Foxc1 and Foxc2 exhibited aberrant expression of Ras regulators, and embryos with LEC-specific deletion of Foxc1 and Foxc2, alone or in combination, exhibited ERK hyperactivation. Pharmacological ERK inhibition in utero abolished the abnormally enlarged lymphatic vessels in FOXC-deficient embryos. Together, these results identify FOXC1 and FOXC2 as essential regulators of lymphangiogenesis and indicate a new potential mechanistic basis for lymphatic-associated diseases.