MicroRNA-409-3p/BTG2 signaling axis improves impaired angiogenesis and wound healing in obese mice.

Wound healing is facilitated by neoangiogenesis, a complex process that is essential to tissue repair in response to injury. MicroRNAs are small, noncoding RNAs that can regulate the wound healing process including stimulation of impaired angiogenesis that is associated with type-2 diabetes (T2D). Expression of miR-409-3p was significantly increased in the nonhealing skin wounds of patients with T2D compared to the non-wounded normal skin, and in the skin of a murine model with T2D. In response to high glucose, neutralization of miR-409-3p markedly improved EC growth and migration in human umbilical vein endothelial cells (HUVECs), promoted wound closure and angiogenesis as measured by increased CD31 in human skin organoids, while overexpression attenuated EC angiogenic responses. Bulk mRNA-Seq transcriptomic profiling revealed BTG2 as a target of miR-409-3p, where overexpression of miR-409-3p significantly decreased BTG2 mRNA and protein expression. A 3' untranslated region (3'-UTR) luciferase assay of BTG2 revealed decreased luciferase activity with overexpression of miR-409-3p, while inhibition had opposite effects. Mechanistically, in response to high glucose, miR-409-3p deficiency in ECs resulted in increased mTOR phosphorylation, meanwhile BTG-anti-proliferation factor 2 (BTG2) silencing significantly decreased mTOR phosphorylation. Endothelial-specific and tamoxifen-inducible miR-409-3p knockout mice (MiR-409IndECKO ) with hyperglycemia that underwent dorsal skin wounding showed significant improvement of wound closure, increased blood flow, granulation tissue thickness (GTT), and CD31 that correlated with increased BTG2 expression. Taken together, our results show that miR-409-3p is a critical mediator of impaired angiogenesis in diabetic skin wound healing.

Src kinase partially mediates cytokine-induced endothelial dysfunction.

OBJECTIVES: Endothelial dysfunction is known to be a key characteristic of preeclampsia (PE) and can contribute to progression of symptoms and injury to multiple organ systems. Delivery is the only treatment for progression of PE, but development of an endothelial-based therapy for PE presents a promising strategy. Growth factors and cytokines are dysregulated in PE and can impact endothelial function, manifesting changes in Ca2+ signaling and interruptions in monolayer barrier function that contribute to symptoms of hypertension, proteinuria, and edema. In this study, we highlight Src kinase as a partial mediator of growth factor and cytokine mediated endothelial dysfunction. STUDY DESIGN: Fura-2 Ca2+ imaging and Electrical Cell Impedance Sensing (ECIS) assays are performed on growth factor or cytokine exposed human umbilical vein endothelial cells (HUVECs). Inhibitors to MEK/ERK (U0126) or Src (PP2) are used to determine the contribution of kinase signaling pathways. MAIN OUTCOME MEASURES: Decreases in HUVEC Ca2+ signaling or monolayer resistance measure endothelial dysfunction. Reversal of endothelial dysfunction by kinase inhibitors reveals the respective contibutions of MEK/ERK and Src kinase. RESULTS: We show that Src inhibition protects Ca2+ signaling responses against insults induced by VEGF165, bFGF, PlGF, TNFα, and IL-1ß. Additionally, we show that Src inhibition protects the endothelial monolayer from the full impact of TNFα insult. Further, we find that MEK/ERK inhibition does not offer protection from growth factor-mediated endothelial dysfunction. CONCLUSIONS: The results of this study suggest cytokine and growth factor-stimulated Src kinase plays a partial role on promoting endothelial dysfunction in HUVECs.

Fatty acids in normal and pathological pregnancies.

Long chain fatty acids, namely omega-3 and omega-6, are essential fatty acids and are necessary for proper pregnancy progression and fetal growth and development. Maternal fatty acid consumption and release of fatty acids from lipid stores provide increased availability of fatty acids for the placenta to transport to the growing fetus. Both omega-3 and omega-6 fatty acids are then utilized for generation of signaling molecules, such as eicosanoids, and for promoting of growth and developmental, most notably in the nervous system. Perturbations in fatty acid concentration and fatty acid signaling have been implicated in three major pregnancy complications - gestational diabetes, preeclampsia, and preterm birth. In this review we discuss the growing literature surrounding the role of fatty acids in normal and pathological pregnancies. Differences in maternal, placental, and fetal fatty acids and molecular regulation of fatty acid signaling and transport are presented. A look into novel fatty acid-based therapies for each of the highlighted disorders are discussed, and may present exciting bench to bedside alternatives to traditional pharmacological intervention.

Cytokine concentrations direct endothelial function in pregnancy and preeclampsia.

Endothelial dysfunction is a prominent feature of preeclampsia, a hypertensive disorder of pregnancy, and contributes to multiple symptoms characteristic of the syndrome. A myriad of growth factors and cytokines are dysregulated in preeclampsia as compared to normal pregnancy, however, a complete appreciation of the effect of changing concentrations of these factors on endothelial function is lacking. In this study, we evaluate the effect of a variety of growth factors and cytokines on Ca2+ signaling and monolayer integrity. We report that VEGF165, TNFα, EGF, and IL-1ß either improve or inhibit Ca2+ signaling depending on dose, whereas TNFα and IL-1ß reduce monolayer integrity and bFGF increases monolayer integrity. Additionally, to model the effects of combinations of growth factors and cytokines, we screened for Ca2+ signaling changes in response to 16 dose combinations of VEGF165 and TNFα together. This revealed an optimal combination capable of supporting pregnancy-adapted Ca2+ signaling, and that changes in either VEGF165 or TNFα dose would result in a shift toward suppressed function. This study shows in detail how growth factor or cytokine concentration effects endothelial cell function. Such data can be used to model how changing growth factor and cytokine levels in normal pregnancy may contribute to healthy endothelial function and in preeclampsia may promote endothelial dysfunction. The results of VEGF165 and TNFα combination treatments suggest that more complex growth factor and cytokine combination modeling may be important in order to more accurately understand the effects of circulating factors on the endothelial function.

Conjugated linoleic acid improves endothelial Ca2+ signaling by blocking growth factor and cytokine-mediated Cx43 phosphorylation.

Sustained Ca2+ burst signaling is crucial for endothelial vasodilator production and is disrupted by growth factors and cytokines. Conjugated linoleic acid (CLA), a Src inhibitor in certain preparations, is generally regarded as safe during pregnancy by the FDA. Multiple CLA preparations; t10, c12 or c9, t11 CLA, or a 1:1 mixture of the two were administered before growth factor or cytokine treatment. Growth factors and cytokines caused a significant decrease in Ca2+ burst numbers in response to ATP stimulation. Both t10, c12 CLA and the 1:1 mixture rescued VEGF165 or TNFα inhibited Ca2+ bursts and correlated with Src-specific phosphorylation of connexin 43. VEGF165, TNFα, and IL-6 in combination at physiologic concentrations revealed IL-6 amplified the inhibitory effects of lower dose of VEGF165 and TNFα. Again, the 1:1 CLA mixture was most effective at rescue of function. Therefore, CLA formulations may be a promising treatment for endothelial dysfunction in diseases such as preeclampsia.

Maternal disease and gasotransmitters.

The three known gasotransmitters, nitric oxide, carbon monoxide, and hydrogen sulfide are involved in key processes throughout pregnancy. Gasotransmitters are known to impact on smooth muscle tone, regulation of immune responses, and oxidative state of cells and their component molecules. Failure of the systems that tightly regulate gasotransmitter production and downstream effects are thought to contribute to common maternal diseases such as preeclampsia and preterm birth. Normal pregnancy-related changes in uterine blood flow depend heavily on gasotransmitter signaling. In preeclampsia, endothelial dysfunction is a major contributor to aberrant gasotransmitter signaling, resulting in hypertension after 20 weeks gestation. Maintenance of pregnancy to term also requires gasotransmitter-mediated uterine quiescence. As the appropriate signals for parturition occur, regulation of gasotransmitter signaling must work in concert with those endocrine signals in order for appropriate labor and delivery timing. Like preeclampsia, preterm birth may have origins in abnormal gasotransmitter signaling. We review the evidence for the involvement of gasotransmitters in preeclampsia and preterm birth, as well as mechanistic and molecular signaling targets.