Arteries and veins have very different susceptibility to certain vascular diseases such as atherosclerosis and vascular calcification. The molecular mechanisms of these differences are not fully understood. In this study, we discovered that COUP-TFII, a transcription factor critical for establishing the venous identity during embryonic vascular development, also regulates the pathophysiological functions of adult blood vessels, especially those directly related to vascular diseases. Specifically, we found that suppression of COUP-TFII in venous ECs switched its phenotype toward pro-atherogenic by up-regulating the expression of inflammatory genes and down-regulating anti-thrombotic genes. ECs with COUP-TFII knockdown also readily undergo endothelial-to-mesenchymal transition (EndoMT) and subsequent osteogenic differentiation with dramatically increased osteogenic transcriptional program and calcium deposition. Consistently, over-expression of COUP-TFII led to the completely opposite effects. In vivo validation of these pro-atherogenic and osteogenic genes also demonstrates a broad consistent differential expression pattern in mouse aorta vs. vena cava ECs, which cannot be explained by the difference in hemodynamic flow. These data reveal phenotypic modulation by different levels of COUP-TFII in arterial and venous ECs, and suggest COUP-TFII may play an important role in the different susceptibilities of arteries and veins to vascular diseases such as atherosclerosis and vascular calcification.

We demonstrate that multivalent, polymeric 8-methoxyguanosine derivatives based on poly(dimethylacrylamide) can enhance the mechanical properties of the low molecular weight hydrogelator 8-methoxy-2′,3′,5′-tri-O-acetylguanosine at biologically relevant salt concentrations. It is proposed that these nongelling polymeric derivatives, under the conditions studied, can result in a significant enhancement of these supramolecular gels (e.g., for gels containing 1 wt % gelator G′ can be increased from ca. 2000 Pa with no additive to 80 000 Pa) by acting as supramolecular cross-linking units. Two competing mechanisms appear to play a role in these cogels. At low polymer concentrations the guanosine-containing polymers tend to act more as solubilizing agents for the gelator, thus weakening the gels, while at high guanosine-containing polymer concentrations the gels show a marked enhancement in mechanical properties consistent with them acting as supramolecular cross-linking agents. As such, the thermomechanical properties of these cogels depend on both the polymer:low molecular weight gelator ratio and the number of 8-methoxyguanosine repeat units present in the polymer additive. Thus, these polymeric guanosine-based additives impart the ability to tailor both the modulus and shear sensitivity of the gels. For example, cogels with a modulus ranging between ca. 95 and 80 000 Pa can be obtained through judicious selection of the type and amount of polymer additive.

Bioactive signals play many important roles on cell function and behavior. In most biological studies, soluble biochemical cues such as growth factors or cytokines are added directly into the media to maintain and/or manipulate cell activities in vitro. However, these methods cannot accurately mimic certain in vivo biological signaling motifs, which are often immobilized to extracellular matrix and also display spatial gradients that are critical for tissue morphology. Besides biochemical cues, biophysical properties such as substrate stiffness can influence cell behavior but is not easy to manipulate under conventional cell culturing practices. Recent development in photocrosslinkable hydrogels provides new tools that allow precise control of spatial biochemical and biophysical cues for biological applications, but doing so requires a comprehensive study on various hydrogel photochemistry kinetics to allow thorough photocrosslink reaction while maintain protein bioactivities at the same time. In this paper, we studied several photochemistry reactions and evaluate key photochemical parameters, such as photoinitiators and ultra-violet (UV) exposure times, to understand their unique contributions to undesired protein damage and cell death. Our data illustrates the retention of protein function and minimize of cell health during photoreactions requires careful selection of photoinitiator type and concentration, and UV exposure times. We also developed a robust method based on thiol-norbornene chemistry for independent control of hydrogel stiffness and spatial bioactive patterns. Overall, we highlight a class of bioactive hydrogels to stiffness control and site specific immobilized bioactive proteins/peptides for the study of cellular behavior such as cellular attraction, repulsion and stem cell fate.

Pluripotent stem cell-derived endothelial cells (ECs) have great potential to be used in vascular therapy or tissue engineering. It is also much desired to obtain arterial or venous ECs for specific applications. Factors that are critical for the proper arterial or venous differentiation from pluripotent stem cells still need to be understood. Here, we aim at investigating this problem deeper by examining neuropilin-1 (Nrp1), an early arterial marker that may be critical for arterial cell fate commitment. Using murine embryonic stem cells as the model system, this study investigates the neuropilin-1 (Nrp1) expression during the differentiation of pluripotent stem cells toward a vascular progenitor population. We hypothesize that Nrp1, an early arterial marker present in a developing embryo, may be more responsive when further induced in vitro toward an arterial fate. We developed a two-step differentiation approach that yielded a large percentage of Nrp1 vascular progenitor cells (VPCs) and investigated their potential to become arterial ECs. We have defined the culture parameters that contribute greatly to the emergence of Nrp1 VPCs: certain soluble factors, especially Wnt and BMP4, early cell-cell contact, and hypoxia. Subsequent isolation of this population demonstrated a highly proliferative and network-forming behavior. The Nrp1 VPCs exhibited increased gene expression of several Notch pathway-related arterial markers compared with Nrp1 VPCs. Most importantly, Nrp1 VPCs demonstrated a dramatically greater response to hemodynamic stimuli by upregulating many arterial markers whereas Nrp1 VPCs have very little response. Surprisingly, these differences between Nrp1 and Nrp1 VPCs are not evident with vascular endothelial growth factor (VEGF) treatment. Our data suggest that Nrp1 VPCs may serve as the arterial progenitor by enhanced response to hemodynamic flow but not to VEGF, whereas Nrp1 VPCs lack the plasticity to become arterial ECs. The findings of this research indicate that Nrp1 VPCs in the murine model act as an important step in the arterial differentiation process.