Preface Endothelial cells lining blood vessel capillaries are not just passive conduits for delivering blood. Tissue-specific endothelium establish specialized vascular niches that deploy specific sets of growth factors, known as angiocrine factors, which actively participate in inducing, specifying, patterning, and guiding organ regeneration and maintaining homeostasis and metabolism. Angiocrine factors upregulated in response to injury orchestrates self-renewal and differentiation of tissue-specific repopulating resident stem and progenitor cells into functional organs. Uncovering the precise mechanisms whereby physiological-levels of angiocrine factors are spatially and temporally produced, and distributed by organotypic endothelium to repopulating cells, will lay the foundation for driving organ repair without scarring.

Summary Chemical or traumatic damage to the liver is frequently associated with aberrant healing(fibrosis) that overrides liver regeneration1–5. The mechanism by which hepatic niche cells differentially modulate regeneration and fibrosis during liver repair remains to be defined6–8. Hepatic vascular niche predominantly represented by liver sinusoidal endothelial cells (LSECs), deploys paracrine trophogens, known as angiocrine factors, to stimulate regeneration9–15. Nevertheless, it remains unknown how pro-regenerative angiocrine signals from LSECs is subverted to promote fibrosis16,17. Here, by combining inducible endothelial cell (EC)-specific mouse gene deletion strategy and complementary models of acute and chronic liver injury, we revealed that divergent angiocrine signals from LSECs elicit regeneration after immediateinjury and provoke fibrosis post chronic insult. The pro-fibrotic transition of vascular niche results from differential expression of stromal derived factor-1 (SDF-1) receptors, CXCR7 and CXCR418–21in LSECs. After acute injury, CXCR7 upregulation in LSECs acts in conjunction with CXCR4 to induce transcription factor Id1, deploying pro-regenerative angiocrine factors and triggering regeneration. Inducible deletion of Cxcr7 in adult mouse LSECs (Cxcr7iΔEC/iΔEC) impaired liver regeneration by diminishing Id1-mediated production of angiocrine factors9–11. By contrast, after chronic injury inflicted by iterative hepatotoxin (carbon tetrachloride) injection and bile duct ligation, constitutive FGFR1 signaling in LSECs counterbalanced CXCR7-dependent pro-regenerative response and augmented CXCR4 expression. This predominance of CXCR4 over CXCR7 expression shifted angiocrine response of LSECs, stimulating proliferation of desmin+hepatic stellate-like cells22,23 and enforcing a pro-fibrotic vascular niche. EC-specific ablation of either Fgfr1 (Fgfr1iΔEC/iΔEC) or Cxcr4 (Cxcr4iΔEC/iΔEC) in mice restored pro-regenerative pathway and prevented FGFR1-mediated maladaptive subversion of angiocrine factors. Similarly, selective CXCR7 activation in LSECs abrogated fibrogenesis. Thus, we have demonstrated 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 fibrosis1,2,4.

SUMMARY 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.

Summary To identify the pathways involved in adult lung regeneration, we have employed left unilateral pneumonectomy (PNX) model that promotes regenerative alveolarization in the remaining intact right lung lobes. Here, we show that PNX stimulates pulmonary capillary endothelial cells (PCECs) to produce paracrine (angiocrine) growth factors that induce proliferation of epithelial progenitor cells supporting alveologenesis. After PNX, endothelial-specific inducible genetic ablation of Vegfr2 and Fgfr1 in mice inhibited production of MMP14 impairing alveolarization. MMP14 via unmasking cryptic EGF-like ectodomain and activation of EGF-receptor (EGFR) expands epithelial progenitor cells. Neutralization of MMP14 impaired EGFR-ligand mediated alveolar regeneration. By contrast, administration of recombinant EGF, or intravascular transplantation of MMP14+ PCECs from wild-type mice, into pneumonectomized Vegfr2/Fgfr1 deficient mice restored alveologenesis and lung inspiratory volume and compliance function. This study shows that VEGFR2 and FGFR1 activation in PCECs by increasing MMP14-dependent bioavailability of EGFR-ligands initiates and sustains alveologenesis and holds promise to develop therapeutic strategies to promote lung regeneration.

Endothelial cells establish an instructive vascular niche that reconstitutes haematopoietic stem and progenitor cells (HSPCs) through release of specific paracrine growth factors, known as angiocrine factors. However, the mechanism by which endothelial cells balance the rate of proliferation and lineage-specific differentiation of HSPCs is unknown. Here, we demonstrate that Akt activation in endothelial cells, through recruitment of mTOR, but not the FoxO pathway, upregulates specific angiocrine factors that support expansion of CD34 − Flt3 − KLS HSPCs with long-term haematopoietic stem cell (LT-HSC) repopulation capacity. Conversely, co-activation of Akt-stimulated endothelial cells with p42/44 MAPK shifts the balance towards maintenance and differentiation of the HSPCs. Selective activation of Akt1 in the endothelial cells of adult mice increased the number of colony forming units in the spleen and CD34 − Flt3 − KLS HSPCs with LT-HSC activity in the bone marrow, accelerating haematopoietic recovery. Therefore, the activation state of endothelial cells modulates reconstitution of HSPCs through the upregulation of angiocrine factors, with Akt-mTOR-activated endothelial cells supporting the self-renewal of LT-HSCs and expansion of HSPCs, whereas MAPK co-activation favours maintenance and lineage-specific differentiation of HSPCs.Acute injury to the bone marrow microenvironment, after treatment with chemotherapy and irradiation, or myelotoxin, suppresses haematopoiesis, which results in the depletion of HSPCs and the development of life-threatening pancytopenias. The interaction of the surviving HSPCs with the bone marrow niche cells rapidly reconstitutes haematopoiesis, rescuing the host from complications associated with long-term bone marrow suppression. Bone marrow niches orchestrate maintenance, expansion and trafficking of HSPCs [1][2][3][4][5] . The osteogenic niche modulates the quiescence of the HSPCs 1-2 , whereas the vascular niche, demarcated by the bone marrow sinusoidal endothelial cells (SECs), regenerates and replenishes the HSPC Results Endothelial cells support both self-renewal and lineage-specific differentiation of HSPCsStudying the role of primary human endothelial cells (PECs) in the regulation of haematopoiesis has been hampered by the need for growthfactor deprivation during culture, which leads to apoptosis of PECs. Supplementation with serum and angiogenic factors, such as VEGF-A and basic-fibroblast growth factor (FGF2), are therefore necessary to maintain PECs for co-culture with HSPCs. However, serum inhibits the self-renewal of HSPCs, whereas FGF2 promotes self-renewal of HSPCs 16 , rendering it difficult to assess the cell-autonomous capacity of PECs to support HSPC homeostasis. To circumvent this problem, PECs can be transduced with an adenovirus gene, early region 4 encoded open reading frame-1 (E4ORF1), which leads to constitutive activation of Akt and enables co-culturing of PECs with HSPCs in serum-and growth factor-free medium for weeks, while maintaining their angio...

SUMMARY During angiogenesis, nascent vascular sprouts fuse to form vascular networks enabling efficient circulation. Mechanisms that stabilize the vascular plexus are not well understood. Sphingosine 1-phosphate (S1P) is a blood-borne lipid mediator implicated in the regulation of vascular and immune systems. Here we describe a mechanism by which the G protein-coupled S1P receptor-1 (S1P1) stabilizes the primary vascular network. A gradient of S1P1 expression from the mature regions of the vascular network to the growing vascular front was observed. In the absence of endothelial S1P1, adherens junctions are destabilized, barrier function is breached, and flow is perturbed resulting in abnormal vascular hypersprouting. Interestingly, S1P1 responds to S1P as well as laminar shear stress to transduce flow-mediated signaling in endothelial cells both in vitro and in vivo. These data demonstrate that blood flow and circulating S1P activate endothelial S1P1 to stabilize blood vessels in development and homeostasis.

ETS transcription factors ETV2, FLI1 and ERG1 specify pluripotent stem cells into endothelial cells (ECs). However, these ECs are unstable and drift towards non-vascular cell fates. We show that human mid-gestation c-Kit− lineage-committed amniotic cells (ACs) can be readily reprogrammed into induced vascular endothelial cells (iVECs). Transient ETV2 expression in ACs generated proliferative but immature iVECs, while co-expression with FLI1/ERG1 endowed iVECs with a vascular repertoire and morphology matching mature stable ECs. Brief TGFβ-inhibition functionalized VEGFR2 signaling, augmenting specification of ACs to iVECs. Genome-wide transcriptional analyses showed that iVECs are similar to adult ECs in which vascular-specific genes are turned on and non-vascular genes are silenced. Functionally, iVECs form long-lasting patent vasculature in Matrigel plugs and regenerating livers. Thus, short-term ETV2 expression and TGFβ-inhibition along with constitutive ERG1/FLI1 co-expression reprogram mature ACs into durable and functional iVECs with clinical-scale expansion potential. Public banking of HLA-typed iVECs would establish a vascular inventory for treatment of genetically diverse disorders.

Summary Tumor endothelial cells (ECs) promote cancer progression in ways beyond their role as conduits supporting metabolism. However, it is not understood how vascular niche-derived paracrine factors, known as angiocrine factors, provoke tumor aggressiveness. Here, we show that FGF4 produced by B-Cell lymphoma cells (LCs) through activating FGFR1 upregulates the Notch-ligand Jagged1 (Jag1) on neighboring tumor ECs. In turn, upregulation of Jag1 on ECs reciprocally induces Notch2-Hey1 in LCs. This crosstalk enforces aggressive CD44+IGF1R+CSF1R+ LC phenotypes, including extra-nodal invasion and chemoresistance. Inducible EC-selective deletion of Fgfr1 or Jag1 in the Eμ-Myc lymphoma model or impairing Notch2 signaling in mouse and human LCs diminished lymphoma aggressiveness and prolonged mouse survival. Thus, targeting the angiocrine FGF4-FGFR1/Jag1-Notch2 loop could inhibit LC aggressiveness and enhance chemosensitivity.