Mark E. Kleinman

Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1

The trafficking of circulating stem and progenitor cells to areas of tissue damage is poorly understood. The chemokine stromal cell–derived factor-1 (SDF-1 or CXCL12) mediates homing of stem cells to bone marrow by binding to CXCR4 on circulating cells. SDF-1 and CXCR4 are expressed in complementary patterns during embryonic organogenesis and guide primordial stem cells to sites of rapid vascular expansion. However, the regulation of SDF-1 and its physiological role in peripheral tissue repair remain incompletely understood. Here we show that SDF-1 gene expression is regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1) in endothelial cells, resulting in selective in vivo expression of SDF-1 in ischemic tissue in direct proportion to reduced oxygen tension. HIF-1-induced SDF-1 expression increases the adhesion, migration and homing of circulating CXCR4-positive progenitor cells to ischemic tissue. Blockade of SDF-1 in ischemic tissue or CXCR4 on circulating cells prevents progenitor cell recruitment to sites of injury. Discrete regions of hypoxia in the bone marrow compartment also show increased SDF-1 expression and progenitor cell tropism. These data show that the recruitment of CXCR4-positive progenitor cells to regenerating tissues is mediated by hypoxic gradients via HIF-1-induced expression of SDF-1.

Sequence- and target-independent angiogenesis suppression by siRNA via TLR3

Clinical trials of small interfering RNA (siRNA) targeting vascular endothelial growth factor-A (VEGFA) or its receptor VEGFR1 (also called FLT1), in patients with blinding choroidal neovascularization (CNV) from age-related macular degeneration, are premised on gene silencing by means of intracellular RNA interference (RNAi). We show instead that CNV inhibition is a siRNA-class effect: 21-nucleotide or longer siRNAs targeting non-mammalian genes, non-expressed genes, non-genomic sequences, pro- and anti-angiogenic genes, and RNAi-incompetent siRNAs all suppressed CNV in mice comparably to siRNAs targeting Vegfa or Vegfr1 without off-target RNAi or interferon-α/β activation. Non-targeted (against non-mammalian genes) and targeted (against Vegfa or Vegfr1) siRNA suppressed CNV via cell-surface toll-like receptor 3 (TLR3), its adaptor TRIF, and induction of interferon-γ and interleukin-12. Non-targeted siRNA suppressed dermal neovascularization in mice as effectively as Vegfa siRNA. siRNA-induced inhibition of neovascularization required a minimum length of 21 nucleotides, a bridging necessity in a modelled 2:1 TLR3–RNA complex. Choroidal endothelial cells from people expressing the TLR3 coding variant 412FF were refractory to extracellular siRNA-induced cytotoxicity, facilitating individualized pharmacogenetic therapy. Multiple human endothelial cell types expressed surface TLR3, indicating that generic siRNAs might treat angiogenic disorders that affect 8% of the world’s population, and that siRNAs might induce unanticipated vascular or immune effects.

Corneal avascularity is due to soluble VEGF receptor-1.

Corneal avascularity—the absence of blood vessels in the cornea—is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.

DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration

Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.

DICER1 Loss and Alu RNA Induce Age-Related Macular Degeneration via the NLRP3 Inflammasome and MyD88

Alu RNA accumulation due to DICER1 deficiency in the retinal pigmented epithelium (RPE) is implicated in geographic atrophy (GA), an advanced form of age-related macular degeneration that causes blindness in millions of individuals. The mechanism of Alu RNA-induced cytotoxicity is unknown. Here we show that DICER1 deficit or Alu RNA exposure activates the NLRP3 inflammasome and triggers TLR-independent MyD88 signaling via IL18 in the RPE. Genetic or pharmacological inhibition of inflammasome components (NLRP3, Pycard, Caspase-1), MyD88, or IL18 prevents RPE degeneration induced by DICER1 loss or Alu RNA exposure. These findings, coupled with our observation that human GA RPE contains elevated amounts of NLRP3, PYCARD, and IL18 and evidence of increased Caspase-1 and MyD88 activation, provide a rationale for targeting this pathway in GA. Our findings also reveal a function of the inflammasome outside the immune system and an immunomodulatory action of mobile elements.

Alternatively spliced vascular endothelial growth factor receptor-2 is an essential endogenous inhibitor of lymphatic vessel growth

Disruption of the precise balance of positive and negative molecular regulators of blood and lymphatic vessel growth can lead to myriad diseases. Although dozens of natural inhibitors of hemangiogenesis have been identified, an endogenous selective inhibitor of lymphatic vessel growth has not to our knowledge been previously described. We report the existence of a splice variant of the gene encoding vascular endothelial growth factor receptor-2 (Vegfr-2) that encodes a secreted form of the protein, designated soluble Vegfr-2 (sVegfr-2), that inhibits developmental and reparative lymphangiogenesis by blocking Vegf-c function. Tissue-specific loss of sVegfr-2 in mice induced, at birth, spontaneous lymphatic invasion of the normally alymphatic cornea and hyperplasia of skin lymphatics without affecting blood vasculature. Administration of sVegfr-2 inhibited lymphangiogenesis but not hemangiogenesis induced by corneal suture injury or transplantation, enhanced corneal allograft survival and suppressed lymphangioma cellular proliferation. Naturally occurring sVegfr-2 thus acts as a molecular uncoupler of blood and lymphatic vessels; modulation of sVegfr-2 might have therapeutic effects in treating lymphatic vascular malformations, transplantation rejection and, potentially, tumor lymphangiogenesis and lymphedema (pages 993–994)

CCR3 is a target for age-related macular degeneration diagnosis and therapy.

Age-related macular degeneration (AMD), a leading cause of blindness worldwide, is as prevalent as cancer in industrialized nations. Most blindness in AMD results from invasion of the retina by choroidal neovascularisation (CNV). Here we show that the eosinophil/mast cell chemokine receptor CCR3 is specifically expressed in choroidal neovascular endothelial cells in humans with AMD, and that despite the expression of its ligands eotaxin-1, -2 and -3, neither eosinophils nor mast cells are present in human CNV. Genetic or pharmacological targeting of CCR3 or eotaxins inhibited injury-induced CNV in mice. CNV suppression by CCR3 blockade was due to direct inhibition of endothelial cell proliferation, and was uncoupled from inflammation because it occurred in mice lacking eosinophils or mast cells, and was independent of macrophage and neutrophil recruitment. CCR3 blockade was more effective at reducing CNV than vascular endothelial growth factor A (VEGF-A) neutralization, which is in clinical use at present, and, unlike VEGF-A blockade, is not toxic to the mouse retina. In vivo imaging with CCR3-targeting quantum dots located spontaneous CNV invisible to standard fluorescein angiography in mice before retinal invasion. CCR3 targeting might reduce vision loss due to AMD through early detection and therapeutic angioinhibition.

Small interfering RNA-induced TLR3 activation inhibits blood and lymphatic vessel growth.

Neovascularization in response to tissue injury consists of the dual invasion of blood (hemangiogenesis) and lymphatic (lymphangiogenesis) vessels. We reported recently that 21-nt or longer small interfering RNAs (siRNAs) can suppress hemangiogenesis in mouse models of choroidal neovascularization and dermal wound healing independently of RNA interference by directly activating Toll-like receptor 3 (TLR3), a double-stranded RNA immune receptor, on the cell surface of blood endothelial cells. Here, we show that a 21-nt nontargeted siRNA suppresses both hemangiogenesis and lymphangiogenesis in mouse models of neovascularization induced by corneal sutures or hindlimb ischemia as efficiently as a 21-nt siRNA targeting vascular endothelial growth factor-A. In contrast, a 7-nt nontargeted siRNA, which is too short to activate TLR3, does not block hemangiogenesis or lymphangiogenesis in these models. Exposure to 21-nt siRNA, which we demonstrate is not internalized unless cell-permeating moieties are used, triggers phosphorylation of cell surface TLR3 on lymphatic endothelial cells and induces apoptosis. These findings introduce TLR3 activation as a method of jointly suppressing blood and lymphatic neovascularization and simultaneously raise new concerns about the undesirable effects of siRNAs on both circulatory systems.

Selective recruitment of endothelial progenitor cells to ischemic tissues with increased neovascularization.

Tissue ischemia remains a common problem in plastic surgery and one for which proangiogenic approaches have been investigated. Given the recent discovery of circulating endothelial stem or progenitor cells that are able to form new blood vessels, the authors sought to determine whether these cells might selectively traffic to regions of tissue ischemia and induce neovascularization. Endothelial progenitor cells were isolated from the peripheral blood of healthy human volunteers and expanded ex vivo for 7 days. Elevation of a cranially based random-pattern skin flap was performed in nude mice, after which they were injected with fluorescent-labeled endothelial progenitor cells (5 × 105; n = 15), fluorescent-labeled human microvascular endothelial cells (5 × 105; n = 15), or media alone (n = 15). Histologic examination demonstrated that endothelial progenitor cells were recruited to ischemic tissue and first appeared by postoperative day 3. Subsequently, endothelial progenitor cell numbers increased exponentially over time for the remainder of the study [0 cells/mm2 at day 0 (n = 3), 9.6 ± 0.9 cells/mm2 at day 3 (n = 3), 24.6 ± 1.5 cells/mm2 at day 7 (n = 3), and 196.3 ± 9.6 cells/mm2 at day 14 (n = 9)]. At all time points, endothelial progenitor cells localized preferentially to ischemic tissue and healing wound edges, and were not observed in normal, uninjured tissues. Endothelial progenitor cell transplantation led to a statistically significant increase in vascular density in ischemic tissues by postoperative day 14 [28.7 ± 1.2 in the endothelial progenitor cell group (n = 9) versus 18 ± 1.1 in the control media group (n = 9) and 17.7 ± 1.0 in the human microvascular endothelial cell group (n = 9; p < 0.01)]. Endothelial progenitor cell transplantation also showed trends toward increased flap survival [171.2 ± 18 mm2 in the endothelial progenitor cell group (n = 12) versus 134.2 ± 10 mm2 in the media group (n = 12) and 145.0 ± 13 mm2 in the human microvascular endothelial cell group (n = 12)], but this did not reach statistical significance. These findings indicate that local tissue is- chemia is a potent stimulus for the recruitment of circulating endothelial progenitor cells. Systemic delivery of endothelial progenitor cells increased neovascularization and suggests that autologous endothelial progenitor cell transplantation may have a role in the salvage of ischemic tissue.

Increased circulating AC133+ CD34+ endothelial progenitor cells in children with hemangioma.

UNLABELLED Hemangioma is the most common soft-tissue tumor of infancy. Despite the frequency of these vascular tumors, the origin of hemangioma-endothelial cells is unknown. Circulating endothelial progenitor cells (EPCs) have recently been identified as vascular stem cells with the capacity to contribute to postnatal vascular development. We have attempted to determine whether circulating EPCs are increased in hemangioma patients and thereby provide insight into the role of EPCs in hemangioma growth. METHODS AND RESULTS Peripheral blood mononuclear cells (PBMCs) were isolated from hemangioma patients undergoing surgical resection (N = 5) and from age-matched controls (N = 5) undergoing strabismus correction surgery. PBMCs were stained with fluorescent-labeled antibodies for AC133, CD34, and VEGFR2/KDR. Fluorescent-labeled isotype antibodies served as negative controls. Histologic sections of surgical specimens were stained with the specific hemangioma markers Glut1, CD32, and merosin, to confirm the diagnosis of common hemangioma of infancy. EPCs harvested from healthy adult volunteers were stained with Glut1, CD32, and merosin, to assess whether cultured EPCs express known hemangioma markers. Hemangioma patients had a 15-fold increase in the number of circulating CD34 AC133 dual-staining cells relative to controls (0.78+/-0.14% vs.0.052+/-0.017%, respectively). Similarly, the number of PBMCs that stained positively for both CD34 and KDR was also increased in hemangioma patients (0.49+/-0.074% vs. 0.19+/-0.041% in controls). Cultured EPCs stained positively for the known hemangioma markers Glut1, CD32, merosin. CONCLUSIONS This is the first study to suggest a role for EPCs in the pathogenesis of hemangioma. Our results imply that increased levels of circulating EPCs may contribute to the formation of this vascular tumor.