Pulmonary arterial hypertension is characterized by vascular remodeling associated with obliteration of pulmonary arterioles and formation of plexiform lesions comprised of hyperproliferative endothelial and vascular smooth muscle cells. Here, we describe a novel, microRNA-dependent association between APLN and FGF2 pathways in the pulmonary artery endothelial cells (PAECs), where disruption of APLN signaling results in a robust increase in FGF2 expression. We show that this link is mediated by two microRNAs, miR-424 and miR-503, that are regulated by APLN and significantly downregulated in PAH. MiR-424 and miR-503 exert anti-proliferative effects by targeting FGF2 and FGFR1. Overexpression of miR-424 and miR-503 in PAECs promoted cellular quiescence and inhibited the capacity of PAEC conditioned media to induce proliferation of pulmonary artery smooth muscle cells. We show that reconstitution of miR-424 and miR-503 can ameliorate pulmonary hypertension in experimental models. These studies demonstrate the importance of APLN-miR-424/503-FGF axis in maintaining pulmonary vascular homeostasis.

Rationale The peptide ligand apelin and its receptor APJ constitute a signaling pathway with numerous effects on the cardiovascular system, including cardiovascular development in model organisms such as xenopus and zebrafish. Objective This study aimed to characterize the embryonic lethal phenotype of the Apj−/− mice and define the involved downstream signaling targets. Methods and Results We report the first characterization of the embryonic lethality of the Apj−/− mice. Greater than half of the expected Apj−/− embryos died in utero due to cardiovascular developmental defects. Those succumbing to early embryonic death had markedly deformed vasculature of the yolk sac and the embryo, as well as poorly looped hearts with aberrantly formed right ventricles and defective atrioventricular cushion formation. Apj−/− embryos surviving to later stages demonstrated incomplete vascular maturation due to a deficiency of vascular smooth muscle cells, and impaired myocardial trabeculation and ventricular wall development. The molecular mechanism implicates a novel, non-canonical signaling pathway downstream of apelin-APJ involving Gα13, which induces histone deacetylase (HDAC) 4 and HDAC5 phosphorylation and cytoplasmic translocation, resulting in activation of MEF2 (myocyte enhancer factor 2). Apj−/− mice have greater endocardial Hdac4 and Hdac5 nuclear localization, and reduced expression of the MEF2 transcriptional target Klf2. We identify a number of commonly shared transcriptional targets among apelin-APJ, Gα13, and MEF2 in endothelial cells, which are significantly decreased in the Apj−/− embryos and endothelial cells. Conclusions Our results demonstrate a novel role for apelin-APJ signaling as a potent regulator of endothelial MEF2 function in the developing cardiovascular system.

The lateral migration of microspheres across streamlines induced by elasticity and inertia in a square microchannel flow of viscoelastic fluids is investigated using a holographic microscopy technique. We experimentally demonstrate the exact particle positions driven by the elasticity of fluid in the channel cross-section. The effects of the blockage ratio, flow rate, and shear-thinning property of the viscoelastic fluids on particle migration are evaluated. In particular, the focusing patterns of microspheres in three-dimensional volume are analyzed under different conditions, namely, dominant inertia, dominant elasticity, and the combined effects of inertia and elasticity. C 2014 AIP Publishing LLC. [http://dx.

Background Pulmonary arterial hypertension (PAH) is a progressive disease of the pulmonary arterioles, characterized by increased pulmonary arterial pressure and right ventricular failure. The etiology of PAH is complex, but aberrant proliferation of the pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells (PASMCs) is thought to play an important role in its pathogenesis. Understanding the mechanisms of transcriptional gene regulation involved in pulmonary vascular homeostasis can provide key insights into potential therapeutic strategies. Methods and Results We demonstrate that the activity of the transcription factor myocyte enhancer factor 2 (MEF2) is significantly impaired in the PAECs derived from subjects with PAH. We identified MEF2 as the key cis-acting factor that regulates expression of a number of transcriptional targets involved in pulmonary vascular homeostasis, including microRNAs 424 and 503, connexins 37, connexin 40, Krűppel Like Factor 2 (KLF2) and KLF4, which were found to be significantly decreased in PAH PAECs. The impaired MEF2 activity in PAH PAECs was mediated by excess nuclear accumulation of two class IIa histone deacetylases (HDACs) that inhibit its function, namely HDAC4 and HDAC5. Selective, pharmacologic inhibition of class IIa HDACs led to restoration of MEF2 activity in PAECs, as demonstrated by increased expression of its transcriptional targets, decreased cell migration and proliferation, and rescue of experimental pulmonary hypertension (PH) models. Conclusions Our results demonstrate that strategies to augment MEF2 activity holds potential therapeutic value in PAH. Moreover, we identify selective HDAC IIa inhibition as a viable alternative approach to avoid the potential adverse effects of broad spectrum HDAC inhibition in PAH.

Differentiation of human pluripotent stem cells (hPSCs) into functional cell types is IntroductionHuman embryonic stem cells (hESCs) derived from an early embryo can self-renew indefinitely and differentiate into a variety of cell types. 1 It has been reported that the "stemness" of hESCs is likely maintained through the harmonious actions of signaling pathway networks. 2 Basic fibroblast growth factor (bFGF) is essential for maintaining the stemness of hESCs by highly activating mitogen-activated protein kinase (MAPK) extracellular signalregulated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling, which plays an important role in the stemness of hESCs. 3 Stemness of hESCs is also supported by bFGF-mediated regulation of transforming growth factor-␤ (TGF-␤) signaling 4 ; activation of the TGF-␤/activin/nodal signaling pathway is required to maintain stemness in cooperation with the FGF signaling pathway, whereas its inhibition results in differentiation of hESCs. 5,6 The effect of Wnt signaling on stemness of hESCs is still controversial. Activation of the Wnt pathway by 6-bromoindirubin-3-oxmie, a specific inhibitor of glycogen synthase-3, sustains the undifferentiated status of hESCs. 7 However, activation of Wnt signaling is insufficient to maintain the undifferentiated status of hESCs, because a canonical Wnt signaling is highly activated during differentiation. 8 The stemness of human induced pluripotent stem cells (hiPSCs), like that of hESCs, seems to be maintained by coordinated networks of signaling molecules, although few differences are observed in the gene expression profile. 9,10 Thus, the stemness and differentiation of hESCs and hiPSCs is regulated by complex networks of signaling pathways. It is likely that the modulation of these signaling pathways can induce differentiation of hESCs into a specialized cell type. In fact, bone morphogenic protein 2/4 (BMP2/4), a member of the TGF␤ superfamily, could differentiate hESCs into trophoblasts, primitive endoderm cells, and mesodermal cells. [11][12][13] However, it has been reported that BMP4 is required for sustaining stemness of mouse ESCs by blocking neural differentiation. 14 hESCs could be differentiated to definitive endoderm cells by activation of activin/nodal signaling and suppression of phosphoinositide 3-kinase (PI3K) signaling. 15 Dual inhibition of SMAD signaling by treatment with Noggin and SB431542 resulted in differentiation of hESCs and hiPSCs into neural cells. 16 Many studies have tried to isolate specialized cell types from spontaneously differentiated cells via formation of hESC-derived embryoid bodies using antibodies against cell-typespecific surface markers. 17,18 However, spontaneous differentiation remains inefficient, in that hPSCs cannot be guided toward a specialized lineage at the initial commitment step.The hPSCs provide a possibility that degenerative or damaged tissues can be replaced with hPSC-derived functional cells. A paucity of number and activity of endothelial progenitor cells is correla...

Fluctuations in flow rate invariably occur in microfluidic devices. This fluidic instability results in a deteriorating performance and the suspension of their unique functions occasionally. In this study, a fluidic-LPF (low pass filter), which is composed of an ACU (air compliance unit) and a FCSP (fluidic channel with high fluidic resistance for sufficient preload), has been proposed for providing the stabilization of hydrodynamic flow in microfluidic devices. To investigate the characteristics of various fluidic networks including our fluidic-LPF, we used a parametric identification method to estimate the time constants via a transient response that was based on a discrete parameter model. In addition, we propose the use of a pulsation index (PI) to quantify the fluctuations in flow rate. We verified the formula for PI derived herein by varying individually both the periods and the air compliance volumes in the ACU, both theoretically and experimentally. We found that the PI depended strongly on either the time constants or the periods of the flow rates at the inlet. Additionally, the normalized differences between the experimental results and the theoretical estimations were less than 6%, which shows that the proposed formula for PI can provide an accurate quantification of the fluctuations in flow, and estimate the parametric effects. Finally, we have successfully demonstrated that our fluidic-LPF can regulate fluctuations in the flow at extremely low flow rates (~ 10 μL h(-1)) and can also control severe fluidic fluctuations (PI = 0.67) with excessively long periods (100 s) via a microfluidic viscometer. We therefore believe that the stabilization of hydrodynamic flow using a fluidic-LPF could be used easily and extensively with a range of microfluidic platforms that require constant flow rates.

The biophysical properties of blood have been considered as promising indices for effectively screening the cardiovascular diseases. In this study, a method for the continuous and simultaneous measurement of the biophysical properties of blood, including viscosity, viscoelasticity, and RBC (red blood cell) aggregation is suggested, using a microfluidic device. The microfluidic device has two inlets (A, B), two outlets (A, B), two identical side channels, and one bridge channel. To sequentially induce steady and transient flows of blood samples, a blood sample is carefully delivered into the inlet (A) at a pulsatile flow rate (Q) (Q = 1 mL h, Q = 0 mL h, T = 240 s). By operating a pinch valve connected to the outlet (A), the blood flow is stopped or passed in the left-lower side channel. Three biophysical properties of the blood sample are quantified by analyzing the flow rate in the left-upper side channel (Q), the image intensity in the left-lower side channel (〈I〉), and the blood-filled width in the right-lower side channel (α). First, based on the modified parallel flow method, the blood viscosity (μ) is measured by analyzing the variation in α. Second, using a discrete fluidic circuit model, the time constant (λ) is evaluated by analyzing temporal variations in Q and 1/(1 - α). Then, the blood elasticity (G) is calculated by assuming the linear Maxwell model (i.e., λ = μ/G). Third, the RBC aggregation is quantified in terms of three parameters (〈I〉, A, and A) obtained by analyzing temporal variations in the image intensity. From the experimental demonstrations using various blood samples, it is concluded that the proposed method has the ability to measure the biophysical properties of blood with consistency, as compared with the previous methods. In the near future, the proposed method will be employed for evaluating variations in the biophysical properties of blood, circulating in the extracorporeal rat bypass loop.

Red blood cell (RBC) aggregation and erythrocyte sedimentation rate (ESR) are considered to be promising biomarkers for effectively monitoring blood rheology at extremely low shear rates. In this study, a microfluidic-based measurement technique is suggested to evaluate RBC aggregation under hematocrit variations due to the continuous ESR. After the pipette tip is tightly fitted into an inlet port, a disposable suction pump is connected to the outlet port through a polyethylene tube. After dropping blood (approximately 0.2 mL) into the pipette tip, the blood flow can be started and stopped by periodically operating a pinch valve. To evaluate variations in RBC aggregation due to the continuous ESR, an EAI (Erythrocyte-sedimentation-rate Aggregation Index) is newly suggested, which uses temporal variations of image intensity. To demonstrate the proposed method, the dynamic characterization of the disposable suction pump is first quantitatively measured by varying the hematocrit levels and cavity volume of the suction pump. Next, variations in RBC aggregation and ESR are quantified by varying the hematocrit levels. The conventional aggregation index (AI) is maintained constant, unrelated to the hematocrit values. However, the EAI significantly decreased with respect to the hematocrit values. Thus, the EAI is more effective than the AI for monitoring variations in RBC aggregation due to the ESR. Lastly, the proposed method is employed to detect aggregated blood and thermally-induced blood. The EAI gradually increased as the concentration of a dextran solution increased. In addition, the EAI significantly decreased for thermally-induced blood. From this experimental demonstration, the proposed method is able to effectively measure variations in RBC aggregation due to continuous hematocrit variations, especially by quantifying the EAI.