Systems Biology Analysis of Temporal Dynamics That Govern Endothelial Response to Cyclic Stretch.

Endothelial cells in vivo are subjected to a wide array of mechanical stimuli, such as cyclic stretch. Notably, a 10% stretch is associated with an atheroprotective endothelial phenotype, while a 20% stretch is associated with an atheroprone endothelial phenotype. Here, a systems biology-based approach is used to present a comprehensive overview of the functional responses and molecular regulatory networks that characterize the transition from an atheroprotective to an atheroprone phenotype in response to cyclic stretch. Using primary human umbilical vein endothelial cells (HUVECs), we determined the role of the equibiaxial cyclic stretch in vitro, with changes to the radius of the magnitudes of 10% and 20%, which are representative of physiological and pathological strain, respectively. Following the transcriptome analysis of next-generation sequencing data, we identified four key endothelial responses to pathological cyclic stretch: cell cycle regulation, inflammatory response, fatty acid metabolism, and mTOR signaling, driven by a regulatory network of eight transcription factors. Our study highlights the dynamic regulation of several key stretch-sensitive endothelial functions relevant to the induction of an atheroprone versus an atheroprotective phenotype and lays the foundation for further investigation into the mechanisms governing vascular pathology. This study has significant implications for the development of treatment modalities for vascular disease.

Specification of fetal liver endothelial progenitors to functional zonated adult sinusoids requires c-Maf induction.

The liver vascular network is patterned by sinusoidal and hepatocyte co-zonation. How intra-liver vessels acquire their hierarchical specialized functions is unknown. We study heterogeneity of hepatic vascular cells during mouse development through functional and single-cell RNA-sequencing. The acquisition of sinusoidal endothelial cell identity is initiated during early development and completed postnatally, originating from a pool of undifferentiated vascular progenitors at E12. The peri-natal induction of the transcription factor c-Maf is a critical switch for the sinusoidal identity determination. Endothelium-restricted deletion of c-Maf disrupts liver sinusoidal development, aberrantly expands postnatal liver hematopoiesis, promotes excessive postnatal sinusoidal proliferation, and aggravates liver pro-fibrotic sensitivity to chemical insult. Enforced c-Maf overexpression in generic human endothelial cells switches on a liver sinusoidal transcriptional program that maintains hepatocyte function. c-Maf represents an inducible intra-organotypic and niche-responsive molecular determinant of hepatic sinusoidal cell identity and lays the foundation for the strategies for vasculature-driven liver repair.

Tracking of Endothelial Cell Migration and Stiffness Measurements Reveal the Role of Cytoskeletal Dynamics.

Cell migration is a complex, tightly regulated multistep process in which cytoskeletal reorganization and focal adhesion redistribution play a central role. Core to both individual and collective migration is the persistent random walk, which is characterized by random force generation and resistance to directional change. We first discuss a model that describes the stochastic movement of ECs and characterizes EC persistence in wound healing. To that end, we pharmacologically disrupted cytoskeletal dynamics, cytochalasin D for actin and nocodazole for tubulin, to understand its contributions to cell morphology, stiffness, and motility. As such, the use of Atomic Force Microscopy (AFM) enabled us to probe the topography and stiffness of ECs, while time lapse microscopy provided observations in wound healing models. Our results suggest that actin and tubulin dynamics contribute to EC shape, compressive moduli, and directional organization in collective migration. Insights from the model and time lapse experiment suggest that EC speed and persistence are directionally organized in wound healing. Pharmacological disruptions suggest that actin and tubulin dynamics play a role in collective migration. Current insights from both the model and experiment represent an important step in understanding the biomechanics of EC migration as a therapeutic target.

Laser scanner for 3D reconstruction of a wound's edge and topology.

PURPOSE: Robotic systems have the potential to overcome inherent limitations of humans and offer substantial advantages to patients including reduction in surgery time. Our group has undertaken the challenge of developing autonomous wound closure system. One of the initial steps is to allow accurate assessment of wound skin topology and wound edge location. We present a vision-laser scanner to generate 3D point cloud for 3D reconstruction of wound's edge and topology. METHODS: When the laser range sensor measures Z coordinate, two encoders installed on the actuators of the gantry robot provide the precision values of X, Y coordinates simultaneously. The 3D point cloud of the wound skin is generated by recordings of X, Y and Z during scanning is performed over wound skin surface. To reduce the scanning time, we exploit a supplementary laser LED to project a regular laser spot on the wound skin surface, which can provide an additional measurement point by incorporating artificial neural network estimation approach. In the meantime, the point cloud of the wound edge can be extracted by detecting if the laser spot is located on the wound edge in the image from 2D camera. RESULTS: The mean absolute error (MAE) and standard deviation (σ) of wound edge are measured in MeshLab environment. The MAE (σ) in X (tangent), Y (tangent), and Z (normal) are 0.32 (0.22) mm, 0.37 (0.34) mm, and 0.61 (0.29) mm, respectively. The experimental results demonstrate that the vision-laser scanner attains high accuracy in determining wound edge location along the tangent of the wound skin. CONCLUSION: A vision-laser scanner is developed for 3D reconstruction of wound's edge and topology. The experimental tests on the different wound models revealed the effectiveness of the vision-laser scanner. The proposed scanner can generate 3D point cloud of the wound skin and its edge simultaneously, and thus significantly improve the accuracy of wound closure in clinical applications.

Morphological characterization of Etv2 vascular explants using fractal analysis and atomic force microscopy.

The rapid engraftment of vascular networks is critical for functional incorporation of tissue explants. However, existing methods for inducing angiogenesis utilize approaches that yield vasculature with poor temporal stability or inadequate mechanical integrity, which reduce their robustness in vivo. The transcription factor Ets variant 2 (Etv2) specifies embryonic hematopoietic and vascular endothelial cell (EC) development, and is transiently reactivated during postnatal vascular regeneration and tumor angiogenesis. This study investigates the role for Etv2 upregulation in forming stable vascular beds both in vitro and in vivo. Control and Etv2+ prototypical fetal-derived human umbilical vein ECs (HUVECs) and adult ECs were angiogenically grown into vascular beds. These vessel beds were characterized using fractal dimension and lacunarity, to quantify their branching complexity and space-filling homogeneity, respectively. Atomic force microscopy (AFM) was used to explore whether greater complexity and homogeneity lead to more mechanically stable vessels. Additionally, markers of EC integrity were used to probe for mechanistic clues. Etv2+ HUVECs exhibit greater branching, vessel density, and structural homogeneity, and decreased stiffness in vitro and in vivo, indicating a greater propensity for stable vessel formation. When co-cultured with colon tumor organoid tissue, Etv2+ HUVECs had decreased fractal dimension and lacunarity compared to Etv2+ HUVECs cultured alone, indicating that vessel density and homogeneity of vessel spacing increased due to the presence of Etv2. This study sets forth the novel concept that fractal dimension, lacunarity, and AFM are as informative as conventional angiogenic measurements, including vessel branching and density, to assess vascular perfusion and stability.

Adaptable haemodynamic endothelial cells for organogenesis and tumorigenesis.

Endothelial cells adopt tissue-specific characteristics to instruct organ development and regeneration1,2. This adaptability is lost in cultured adult endothelial cells, which do not vascularize tissues in an organotypic manner. Here, we show that transient reactivation of the embryonic-restricted ETS variant transcription factor 2 (ETV2)3 in mature human endothelial cells cultured in a serum-free three-dimensional matrix composed of a mixture of laminin, entactin and type-IV collagen (LEC matrix) 'resets' these endothelial cells to adaptable, vasculogenic cells, which form perfusable and plastic vascular plexi. Through chromatin remodelling, ETV2 induces tubulogenic pathways, including the activation of RAP1, which promotes the formation of durable lumens4,5. In three-dimensional matrices-which do not have the constraints of bioprinted scaffolds-the 'reset' vascular endothelial cells (R-VECs) self-assemble into stable, multilayered and branching vascular networks within scalable microfluidic chambers, which are capable of transporting human blood. In vivo, R-VECs implanted subcutaneously in mice self-organize into durable pericyte-coated vessels that functionally anastomose to the host circulation and exhibit long-lasting patterning, with no evidence of malformations or angiomas. R-VECs directly interact with cells within three-dimensional co-cultured organoids, removing the need for the restrictive synthetic semipermeable membranes that are required for organ-on-chip systems, therefore providing a physiological platform for vascularization, which we call 'Organ-On-VascularNet'. R-VECs enable perfusion of glucose-responsive insulin-secreting human pancreatic islets, vascularize decellularized rat intestines and arborize healthy or cancerous human colon organoids. Using single-cell RNA sequencing and epigenetic profiling, we demonstrate that R-VECs establish an adaptive vascular niche that differentially adjusts and conforms to organoids and tumoroids in a tissue-specific manner. Our Organ-On-VascularNet model will permit metabolic, immunological and physiochemical studies and screens to decipher the crosstalk between organotypic endothelial cells and parenchymal cells for identification of determinants of endothelial cell heterogeneity, and could lead to advances in therapeutic organ repair and tumour targeting.

Molecular determinants of nephron vascular specialization in the kidney.

Although kidney parenchymal tissue can be generated in vitro, reconstructing the complex vasculature of the kidney remains a daunting task. The molecular pathways that specify and sustain functional, phenotypic and structural heterogeneity of the kidney vasculature are unknown. Here, we employ high-throughput bulk and single-cell RNA sequencing of the non-lymphatic endothelial cells (ECs) of the kidney to identify the molecular pathways that dictate vascular zonation from embryos to adulthood. We show that the kidney manifests vascular-specific signatures expressing defined transcription factors, ion channels, solute transporters, and angiocrine factors choreographing kidney functions. Notably, the ontology of the glomerulus coincides with induction of unique transcription factors, including Tbx3, Gata5, Prdm1, and Pbx1. Deletion of Tbx3 in ECs results in glomerular hypoplasia, microaneurysms and regressed fenestrations leading to fibrosis in subsets of glomeruli. Deciphering the molecular determinants of kidney vascular signatures lays the foundation for rebuilding nephrons and uncovering the pathogenesis of kidney disorders.

Laminar shear stress modulates endothelial luminal surface stiffness in a tissue-specific manner.

OBJECTIVE: Endothelial cells form vascular beds in all organs and are exposed to a range of mechanical forces that regulate cellular phenotype. We sought to determine the role of endothelial luminal surface stiffness in tissue-specific mechanotransduction of laminar shear stress in microvascular mouse cells and the role of arachidonic acid in mediating this response. METHODS: Microvascular mouse endothelial cells were subjected to laminar shear stress at 4 dynes/cm2 for 12 hours in parallel plate flow chambers that enabled real-time optical microscopy and atomic force microscopy measurements of cell stiffness. RESULTS: Lung endothelial cells aligned parallel to flow, while cardiac endothelial cells did not. This rapid alignment was accompanied by increased cell stiffness. The addition of arachidonic acid to cardiac endothelial cells increased alignment and stiffness in response to shear stress. Inhibition of arachidonic acid in lung endothelial cells and embryonic stem cell-derived endothelial cells prevented cellular alignment and decreased cell stiffness. CONCLUSIONS: Our findings suggest that increased endothelial luminal surface stiffness in microvascular cells may facilitate mechanotransduction and alignment in response to laminar shear stress. Furthermore, the arachidonic acid pathway may mediate this tissue-specific process. An improved understanding of this response will aid in the treatment of organ-specific vascular disease.

Endothelial jagged-2 sustains hematopoietic stem and progenitor reconstitution after myelosuppression.

Angiocrine factors, such as Notch ligands, supplied by the specialized endothelial cells (ECs) within the bone marrow and splenic vascular niche play an essential role in modulating the physiology of adult hematopoietic stem and progenitor cells (HSPCs). However, the relative contribution of various Notch ligands, specifically jagged-2, to the homeostasis of HSPCs is unknown. Here, we show that under steady state, jagged-2 is differentially expressed in tissue-specific vascular beds, but its expression is induced in hematopoietic vascular niches after myelosuppressive injury. We used mice with EC-specific deletion of the gene encoding jagged-2 (Jag2) to demonstrate that while EC-derived jagged-2 was dispensable for maintaining the capacity of HSPCs to repopulate under steady-state conditions, by activating Notch2 it did contribute to the recovery of HSPCs in response to myelosuppressive conditions. Engraftment and/or expansion of HSPCs was dependent on the expression of endothelial-derived jagged-2 following myeloablation. Additionally, jagged-2 expressed in bone marrow ECs regulated HSPC cell cycle and quiescence during regeneration. Endothelial-deployed jagged-2 triggered Notch2/Hey1, while tempering Notch2/Hes1 signaling in HSPCs. Collectively, these data demonstrate that EC-derived jagged-2 activates Notch2 signaling in HSPCs to promote hematopoietic recovery and has potential as a therapeutic target to accelerate balanced hematopoietic reconstitution after myelosuppression.

Endothelial transplantation rejuvenates aged hematopoietic stem cell function.

Age-related changes in the hematopoietic compartment are primarily attributed to cell-intrinsic alterations in hematopoietic stem cells (HSCs); however, the contribution of the aged microenvironment has not been adequately evaluated. Understanding the role of the bone marrow (BM) microenvironment in supporting HSC function may prove to be beneficial in treating age-related functional hematopoietic decline. Here, we determined that aging of endothelial cells (ECs), a critical component of the BM microenvironment, was sufficient to drive hematopoietic aging phenotypes in young HSCs. We used an ex vivo hematopoietic stem and progenitor cell/EC (HSPC/EC) coculture system as well as in vivo EC infusions following myelosuppressive injury in mice to demonstrate that aged ECs impair the repopulating activity of young HSCs and impart a myeloid bias. Conversely, young ECs restored the repopulating capacity of aged HSCs but were unable to reverse the intrinsic myeloid bias. Infusion of young, HSC-supportive BM ECs enhanced hematopoietic recovery following myelosuppressive injury and restored endogenous HSC function in aged mice. Coinfusion of young ECs augmented aged HSC engraftment and enhanced overall survival in lethally irradiated mice by mitigating damage to the BM vascular microenvironment. These data lay the groundwork for the exploration of EC therapies that can serve as adjuvant modalities to enhance HSC engraftment and accelerate hematopoietic recovery in the elderly population following myelosuppressive regimens.

Concerted regulation of retinal pigment epithelium basement membrane and barrier function by angiocrine factors.

The outer blood-retina barrier is established through the coordinated terminal maturation of the retinal pigment epithelium (RPE), fenestrated choroid endothelial cells (ECs) and Bruch's membrane, a highly organized basement membrane that lies between both cell types. Here we study the contribution of choroid ECs to this process by comparing their gene expression profile before (P5) and after (P30) the critical postnatal period when mice acquire mature visual function. Transcriptome analyses show that expression of extracellular matrix-related genes changes dramatically over this period. Co-culture experiments support the existence of a novel regulatory pathway: ECs secrete factors that remodel RPE basement membrane, and integrin receptors sense these changes triggering Rho GTPase signals that modulate RPE tight junctions and enhance RPE barrier function. We anticipate our results will spawn a search for additional roles of choroid ECs in RPE physiology and disease.

PEG-Immobilized Keratin for Protein Drug Sequestration and pH-Mediated Delivery.

Protein drugs like growth factors are promising therapeutics for damaged-tissue repair. Their local delivery often requires biomaterial carriers for achieving the therapeutic dose range while extending efficacy. In this study, polyethylene glycol (PEG) and keratin were crosslinked and used as sponge-like scaffolds (KTN-PEG) to absorb test proteins with different isoelectric points (pI): albumin (~5), hemoglobin (~7), and lysozyme (~11). The protein release kinetics was influenced by charge at physiological pH 7.4. The keratin network, with pI 5.3, electrostatically attracted lysozyme and repulsed albumin generating the release rate profile: albumin > hemoglobin > lysozyme. However, under acidic conditions (pH 4), all proteins including keratins were positively charged and consequently intermolecular repulsion altered the release hierarchy, now determined by size (MW) diffusion: lysozyme (14 kDa) > hemoglobin (64 kDa) > albumin (66 kDa). Vascular endothelial growth factor C (VEGF-C), with properties comparable to lysozyme, was absorbed into the KTN-PEG scaffold. Endothelial cells cultured on this substrate had significantly larger numbers than on scaffolds without VEGF-C suggesting that the ionically bound and retained growth factor at neutral pH indirectly increased acute cell attachment and viability. PEG and keratin based sequestrations of proteins with basic pIs are therefore a feasible strategy with potential applications for selective biologics delivery.

Targeting of the pulmonary capillary vascular niche promotes lung alveolar repair and ameliorates fibrosis.

Although the lung can undergo self-repair after injury, fibrosis in chronically injured or diseased lungs can occur at the expense of regeneration. Here we study how a hematopoietic-vascular niche regulates alveolar repair and lung fibrosis. Using intratracheal injection of bleomycin or hydrochloric acid in mice, we show that repetitive lung injury activates pulmonary capillary endothelial cells (PCECs) and perivascular macrophages, impeding alveolar repair and promoting fibrosis. Whereas the chemokine receptor CXCR7, expressed on PCECs, acts to prevent epithelial damage and ameliorate fibrosis after a single round of treatment with bleomycin or hydrochloric acid, repeated injury leads to suppression of CXCR7 expression and recruitment of vascular endothelial growth factor receptor 1 (VEGFR1)-expressing perivascular macrophages. This recruitment stimulates Wnt/ß-catenin-dependent persistent upregulation of the Notch ligand Jagged1 (encoded by Jag1) in PCECs, which in turn stimulates exuberant Notch signaling in perivascular fibroblasts and enhances fibrosis. Administration of a CXCR7 agonist or PCEC-targeted Jag1 shRNA after lung injury promotes alveolar repair and reduces fibrosis. Thus, targeting of a maladapted hematopoietic-vascular niche, in which macrophages, PCECs and perivascular fibroblasts interact, may help to develop therapy to spur lung regeneration and alleviate fibrosis.

A Parallel-Plate Flow Chamber for Mechanical Characterization of Endothelial Cells Exposed to Laminar Shear Stress.

Shear stresses induced by laminar fluid flow are essential to properly recapitulate the physiological microenvironment experienced by endothelial cells (ECs). ECs respond to these stresses via mechanotransduction by modulating their phenotype and biomechanical characteristics, which can be characterized by Atomic Force Microscopy (AFM). Parallel Plate Flow Chambers (PPFCs) apply unidirectional laminar fluid flow to EC monolayers in vitro. Since ECs in sealed PPFCs are inaccessible to AFM probes, cone-and-plate viscometers (CPs) are commonly used to apply shear stress. This paper presents a comparison of the efficacies of both methods. Computational Fluid Dynamic simulation and validation testing using EC responses as a metric have indicated limitations in the use of CPs to apply laminar shear stress. Monolayers subjected to laminar fluid flow in a PPFC respond by increasing cortical stiffness, elongating, and aligning filamentous actin in the direction of fluid flow to a greater extent than CP devices. Limitations using CP devices to provide laminar flow across an EC monolayer suggest they are better suited when studying EC response for disturbed flow conditions. PPFC platforms allow for exposure of ECs to laminar fluid flow conditions, recapitulating cellular biomechanical behaviors, whereas CP platforms allow for mechanical characterization of ECs under secondary flow.

Platelet-derived SDF-1 primes the pulmonary capillary vascular niche to drive lung alveolar regeneration.

The lung alveoli regenerate after surgical removal of the left lobe by pneumonectomy (PNX). How this alveolar regrowth/regeneration is initiated remains unknown. We found that platelets trigger lung regeneration by supplying stromal-cell-derived factor-1 (SDF-1, also known as CXCL12). After PNX, activated platelets stimulate SDF-1 receptors CXCR4 and CXCR7 on pulmonary capillary endothelial cells (PCECs) to deploy the angiocrine membrane-type metalloproteinase MMP14, stimulating alveolar epithelial cell (AEC) expansion and neo-alveolarization. In mice lacking platelets or platelet Sdf1, PNX-induced alveologenesis was diminished. Reciprocally, infusion of Sdf1(+/+) but not Sdf1-deficient platelets rescued lung regeneration in platelet-depleted mice. Endothelial-specific ablation of Cxcr4 and Cxcr7 in adult mice similarly impeded lung regeneration. Notably, mice with endothelial-specific Mmp14 deletion exhibited impaired expansion of AECs but not PCECs after PNX, which was not rescued by platelet infusion. Therefore, platelets prime PCECs to initiate lung regeneration, extending beyond their haemostatic contribution. Therapeutic targeting of this haemo-vascular niche could enable regenerative therapy for lung diseases.

Divergent angiocrine signals from vascular niche balance liver regeneration and fibrosis.

Chemical or traumatic damage to the liver is frequently associated with aberrant healing (fibrosis) that overrides liver regeneration. The mechanism by which hepatic niche cells differentially modulate regeneration and fibrosis during liver repair remains to be defined. Hepatic vascular niche predominantly represented by liver sinusoidal endothelial cells deploys paracrine trophogens, known as angiocrine factors, to stimulate regeneration. Nevertheless, it is not known how pro-regenerative angiocrine signals from liver sinusoidal endothelial cells is subverted to promote fibrosis. Here, by combining an inducible endothelial-cell-specific mouse gene deletion strategy and complementary models of acute and chronic liver injury, we show that divergent angiocrine signals from liver sinusoidal endothelial cells stimulate regeneration after immediate injury and provoke fibrosis after chronic insult. The pro-fibrotic transition of vascular niche results from differential expression of stromal-derived factor-1 receptors, CXCR7 and CXCR4 (refs 18, 19, 20, 21), in liver sinusoidal endothelial cells. After acute injury, CXCR7 upregulation in liver sinusoidal endothelial cells acts with CXCR4 to induce transcription factor Id1, deploying pro-regenerative angiocrine factors and triggering regeneration. Inducible deletion of Cxcr7 in sinusoidal endothelial cells (Cxcr7(iΔEC/iΔEC)) from the adult mouse liver impaired liver regeneration by diminishing Id1-mediated production of angiocrine factors. By contrast, after chronic injury inflicted by iterative hepatotoxin (carbon tetrachloride) injection and bile duct ligation, constitutive FGFR1 signalling in liver sinusoidal endothelial cells counterbalanced CXCR7-dependent pro-regenerative response and augmented CXCR4 expression. This predominance of CXCR4 over CXCR7 expression shifted angiocrine response of liver sinusoidal endothelial cells, stimulating proliferation of desmin(+) hepatic stellate-like cells and enforcing a pro-fibrotic vascular niche. Endothelial-cell-specific ablation of either Fgfr1 (Fgfr1(iΔEC/iΔEC)) or Cxcr4 (Cxcr4(iΔEC/iΔEC)) in mice restored the pro-regenerative pathway and prevented FGFR1-mediated maladaptive subversion of angiocrine factors. Similarly, selective CXCR7 activation in liver sinusoidal endothelial cells abrogated fibrogenesis. Thus, we demonstrate that in response to liver injury, differential recruitment of pro-regenerative CXCR7-Id1 versus pro-fibrotic FGFR1-CXCR4 angiocrine pathways in vascular niche balances regeneration and fibrosis. These results provide a therapeutic roadmap to achieve hepatic regeneration without provoking fibrosis.

Scaffold biomaterials for nano-pathophysiology.

This review is intended to provide an overview of tissue engineering strategies using scaffold biomaterials to develop a vascularized tissue engineered construct for nano-pathophysiology. Two primary topics are discussed. The first is the biological or synthetic microenvironments that regulate cell behaviors in pathological conditions and tissue regeneration. Second is the use of scaffold biomaterials with angiogenic factors and/or cells to realize vascularized tissue engineered constructs for nano-pathophysiology. These topics are significantly overlapped in terms of three-dimensional (3-D) geometry of cells and blood vessels. Therefore, this review focuses on neovascularization of 3-D scaffold biomaterials induced by angiogenic factors and/or cells. The novel strategy of this approach in nano-pathophysiology is to utilize the vascularized tissue engineered construct as a tissue model to predict the distribution and subsequent therapeutic efficacy of a drug delivery system with different physicochemical and biological properties.

Akt suppression of TGFß signaling contributes to the maintenance of vascular identity in embryonic stem cell-derived endothelial cells.

The ability to generate and maintain stable in vitro cultures of mouse endothelial cells (ECs) has great potential for genetic dissection of the numerous pathologies involving vascular dysfunction as well as therapeutic applications. However, previous efforts at achieving sustained cultures of primary stable murine vascular cells have fallen short, and the cellular requirements for EC maintenance in vitro remain undefined. In this study, we have generated vascular ECs from mouse embryonic stem (ES) cells and show that active Akt is essential to their survival and propagation as homogeneous monolayers in vitro. These cells harbor the phenotypical, biochemical, and functional characteristics of ECs and expand throughout long-term cultures, while maintaining their angiogenic capacity. Moreover, Akt-transduced embryonic ECs form functional perfused vessels in vivo that anastomose with host blood vessels. We provide evidence for a novel function of Akt in stabilizing EC identity, whereby the activated form of the protein protects mouse ES cell-derived ECs from TGFß-mediated transdifferentiation by downregulating SMAD3. These findings identify a role for Akt in regulating the developmental potential of ES cell-derived ECs and demonstrate that active Akt maintains endothelial identity in embryonic ECs by interfering with active TGFß-mediated processes that would ordinarily usher these cells to alternate fates.

Molecular signatures of tissue-specific microvascular endothelial cell heterogeneity in organ maintenance and regeneration.

Microvascular endothelial cells (ECs) within different tissues are endowed with distinct but as yet unrecognized structural, phenotypic, and functional attributes. We devised EC purification, cultivation, profiling, and transplantation models that establish tissue-specific molecular libraries of ECs devoid of lymphatic ECs or parenchymal cells. These libraries identify attributes that confer ECs with their organotypic features. We show that clusters of transcription factors, angiocrine growth factors, adhesion molecules, and chemokines are expressed in unique combinations by ECs of each organ. Furthermore, ECs respond distinctly in tissue regeneration models, hepatectomy, and myeloablation. To test the data set, we developed a transplantation model that employs generic ECs differentiated from embryonic stem cells. Transplanted generic ECs engraft into regenerating tissues and acquire features of organotypic ECs. Collectively, we demonstrate the utility of informational databases of ECs toward uncovering the extravascular and intrinsic signals that define EC heterogeneity. These factors could be exploited therapeutically to engineer tissue-specific ECs for regeneration.