Characterization of a resident population of adventitial macrophage progenitor cells in postnatal vasculature.

RATIONALE: Macrophages regulate blood vessel structure and function in health and disease. The origins of tissue macrophages are diverse, with evidence for local production and circulatory renewal. OBJECTIVE: We identified a vascular adventitial population containing macrophage progenitor cells and investigated their origins and fate. METHODS AND RESULTS: Single-cell disaggregates from adult C57BL/6 mice were prepared from different tissues and tested for their capacity to form hematopoietic colony-forming units. Aorta showed a unique predilection for generating macrophage colony-forming units. Aortic macrophage colony-forming unit progenitors coexpressed stem cell antigen-1 and CD45 and were adventitially located, where they were the predominant source of proliferating cells in the aortic wall. Aortic Sca-1(+)CD45(+) cells were transcriptionally and phenotypically distinct from neighboring cells lacking stem cell antigen-1 or CD45 and contained a proliferative (Ki67(+)) Lin(-)c-Kit(+)CD135(-)CD115(+)CX3CR1(+)Ly6C(+)CD11b(-) subpopulation, consistent with the immunophenotypic profile of macrophage progenitors. Adoptive transfer studies revealed that Sca-1(+)CD45(+) adventitial macrophage progenitor cells were not replenished via the circulation from bone marrow or spleen, nor was their prevalence diminished by depletion of monocytes or macrophages by liposomal clodronate treatment or genetic deficiency of macrophage colony-stimulating factor. Rather adventitial macrophage progenitor cells were upregulated in hyperlipidemic ApoE(-/-) and LDL-R(-/-) mice, with adventitial transfer experiments demonstrating their durable contribution to macrophage progeny particularly in the adventitia, and to a lesser extent the atheroma, of atherosclerotic carotid arteries. CONCLUSIONS: The discovery and characterization of resident vascular adventitial macrophage progenitor cells provides new insight into adventitial biology and its participation in atherosclerosis and provokes consideration of the broader existence of local macrophage progenitors in other tissues.

Tissue factor pathway inhibitor blocks angiogenesis via its carboxyl terminus.

OBJECTIVE: Tissue factor pathway inhibitor (TFPI) is the primary regulator of the tissue factor (TF) coagulation pathway. As such, TFPI may regulate the proangiogenic effects of TF. TFPI may also affect angiogenesis independently of TF, through sequences within its polybasic carboxyl terminus (TFPI C terminus [TFPIct]). We aimed to determine the effects of TFPI on angiogenesis and the role of TFPIct. METHODS AND RESULTS: Transgenic overexpression of TFPI attenuated angiogenesis in the murine hindlimb ischemia model and an aortic sprout assay. In vitro, TFPI inhibited endothelial cell migration. Peptides within the human TFPIct inhibited endothelial cell cord formation and migration in response to vascular endothelial growth factor (VEGF) 165 but not VEGF121. Furthermore, exposure to human TFPIct inhibited the phosphorylation of VEGF receptor 2 at residue Lys951, a residue known to be critical for endothelial cell migration. Finally, systemic delivery of a murine TFPIct peptide inhibited angiogenesis in the hindlimb model. CONCLUSION: These data demonstrate an inhibitory role for TFPI in angiogenesis that is, in part, mediated through peptides within its carboxyl terminus. In addition to its known role as a TF antagonist, TFPI, via its carboxyl terminus, may regulate angiogenesis by directly blocking VEGF receptor 2 activation and attenuating the migratory capacity of endothelial cells.

Identification of a monocyte-predisposed hierarchy of hematopoietic progenitor cells in the adventitia of postnatal murine aorta.

BACKGROUND: Hematopoiesis originates from the dorsal aorta during embryogenesis. Although adult blood vessels harbor progenitor populations for endothelial and smooth muscle cells, it is not known if they contain hematopoietic progenitor or stem cells. Here, we hypothesized that the arterial wall is a source of hematopoietic progenitor and stem cells in postnatal life. METHODS AND RESULTS: Single-cell aortic disaggregates were prepared from adult chow-fed C57BL/6 and apolipoprotein E-null (ApoE(-/-)) mice. In short- and long-term methylcellulose-based culture, aortic cells generated a broad spectrum of multipotent and lineage-specific hematopoietic colony-forming units, with a preponderance of macrophage colony-forming units. This clonogenicity was higher in lesion-free ApoE(-/-) mice and localized primarily to stem cell antigen-1-positive cells in the adventitia. Expression of stem cell antigen-1 in the aorta colocalized with canonical hematopoietic stem cell markers, as well as CD45 and mature leukocyte antigens. Adoptive transfer of labeled aortic cells from green fluorescent protein transgenic donors to irradiated C57BL/6 recipients confirmed the content of rare hematopoietic stem cells (1 per 4 000 000 cells) capable of self-renewal and durable, low-level reconstitution of leukocytes. Moreover, the predominance of long-term macrophage precursors was evident by late recovery of green fluorescent protein-positive colonies from recipient bone marrow and spleen that were exclusively macrophage colony-forming units. Although trafficking from bone marrow was shown to replenish some of the hematopoietic potential of the aorta after irradiation, the majority of macrophage precursors appeared to arise locally, suggesting long-term residence in the vessel wall. CONCLUSIONS: The postnatal murine aorta contains rare multipotent hematopoietic progenitor/stem cells and is selectively enriched with stem cell antigen-1-positive monocyte/macrophage precursors. These populations may represent novel, local vascular sources of inflammatory cells.

Tissue factor pathway inhibitor overexpression inhibits hypoxia-induced pulmonary hypertension.

Pulmonary hypertension (PH) is a commonly recognized complication of chronic respiratory disease. Enhanced vasoconstriction, pulmonary vascular remodeling, and in situ thrombosis contribute to the increased pulmonary vascular resistance observed in PH associated with hypoxic lung disease. The tissue factor pathway regulates fibrin deposition in response to acute and chronic vascular injury. We hypothesized that inhibition of the tissue factor pathway would result in attenuation of pathophysiologic parameters typically associated with hypoxia-induced PH. We tested this hypothesis using a chronic hypoxia-induced murine model of PH using mice that overexpress tissue factor pathway inhibitor (TFPI) via the smooth muscle-specific promoter SM22 (TFPI(SM22)). TFPI(SM22) mice have increased pulmonary TFPI expression compared with wild-type (WT) mice. In WT mice, exposure to chronic hypoxia (28 d at 10% O(2)) resulted in increased systolic right ventricular and mean pulmonary arterial pressures, changes that were significantly reduced in TFPI(SM22) mice. Chronic hypoxia also resulted in significant pulmonary vascular muscularization in WT mice, which was significantly reduced in TFPI(SM22) mice. Given the pleiotropic effects of TFPI, autocrine and paracrine mechanisms for these hemodynamic effects were considered. TFPI(SM22) mice had less pulmonary fibrin deposition than WT mice at 3 days after exposure to hypoxia, which is consistent with the antithrombotic effects of TFPI. Additionally, TFPI(SM22) mice had a significant reduction in the number of proliferating (proliferating cell nuclear antigen positive) pulmonary vascular smooth muscle cells compared with WT mice, which is consistent with in vitro findings. These findings demonstrate that overexpression of TFPI results in improved hemodynamic performance and reduced pulmonary vascular remodeling in a murine model of hypoxia-induced PH. This improvement is in part due to the autocrine and paracrine effects of TFPI overexpression.

The effect of vascular smooth muscle cell-targeted expression of tissue factor pathway inhibitor in a murine model of arterial thrombosis.

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type protease inhibitor that regulates the extrinsic pathway of coagulation by inhibiting the factor VIIa/tissue factor (TF) catalytic complex. TFPI is expressed by both endothelial and smooth muscle cells in the vasculature and circulates at low levels. The role of local vascular TFPI in thrombosis and the development of vascular disease is unknown. To establish an experimental animal model to directly modulate smooth muscle cell-derived TFPI on the development of arterial thrombosis, transgenic mice in which a cDNA encoding murine TFPI is expressed from the murine SM22alpha promoter were generated. Expression of transgenic mRNA was 4-fold higher than the level of endogenous TFPI mRNA in arteries from transgenic mice. In situ hybridization confirmed that expression of the transgene was limited to medial vascular smooth muscle cells. Vascular TFPI activity was increased to 2 to 3-fold in carotid homogenates. There was no difference in plasma TFPI levels or hemostatic measures (PT, aPTT and tail vein bleeding times) between these mice and their wildtype littermates. In a ferric chloride-induced model of carotid thrombosis, homozygotic transgenic mice demonstrated resistance to thrombotic occlusion compared to wildtype littermates. In transgenic mice 22% occluded within 30 minutes of application while 84% of wild type mice occluded within the same time frame (p<0.01). Heterozygotic transgenic mice had an intermediate thrombotic phenotype. Taken together, these data indicated that local VSMC-specific TFPI overexpression attenuated ferric chloride-induced thrombosis without systemic or hemostatic effects. Furthermore, this transgenic mouse model should prove useful for studying the role of TFPI in the development and progression of vascular disease.

Gene transfer of a novel vasoactive natriuretic peptide stimulates cGMP and lowers blood pressure in mice.

Dendroaspis natriuretic peptide (DNP) is a recently described peptide produced by Dendroaspis angusticeps with structural and functional similarities to mammalian natriuretic peptides. These similarities suggest a potential role for DNP in cardiovascular therapeutics. To determine the physiological effects of chronic delivery of DNP, a gene transfer approach using first generation adenoviral vectors was utilized. Although the gene for DNP has not been cloned in any species, the peptide sequence in the snake is known. Preferred mammalian codons for snake DNP were cloned downstream of either the leader sequence (referred to as pBDNP-1) or prepropeptide sequence of human brain natriuretic peptide (BNP) cDNA (referred to as pBDNP-2). Transfections with pBDNP-1 or pBDNP-2 resulted in expected forms of chimeric DNP (cDNP) in cell lysates and conditioned media. Functional studies demonstrated the ability of both forms of cDNP within conditioned media to stimulate cGMP production in human vascular smooth muscle cells (hVSMC). Expressed cDNP inhibited hVSMC proliferation and stimulated vasorelaxation in a similar fashion. To investigate the chronic physiological effects of administration of cDNP, an adenoviral vector expressing cDNP (Ad-BDNP) was generated. Intravenous delivery of Ad-BDNP in mice resulted in dose-dependent systemic expression of cDNP. The highest level of expression was associated with consistent elevation of its presumed second messenger (cGMP) for 21 days but with transient lowering of systolic blood pressure in normotensive mice. This study demonstrates the biological features of the expression of the xenogenic peptide DNP.

Autologous culture-modified mononuclear cells confer vascular protection after arterial injury.

BACKGROUND: Bone marrow-derived cells have been shown to contribute to endothelial replacement after vascular injury. In vitro culture of peripheral blood mononuclear cells produces cells with phenotypic characteristics of endothelium. To test the hypothesis that delivery of autologous culture-modified mononuclear cells (CMMCs) to injured arteries could attenuate the vascular response to injury, a rabbit model was studied. METHODS AND RESULTS: Rabbit peripheral blood mononuclear cells were cultured in endothelial growth media for 7 to 12 days, yielding highly proliferative cells with distinct endothelial phenotype (expressing CD31 and endothelial nitric oxide synthase and capable of acetylated LDL uptake). A rabbit model of balloon carotid injury was used to evaluate the effect of day 7 CMMC delivery on vascular responses. Animals underwent balloon injury and immediate delivery of autologous CMMCs or buffered saline by 20 minutes of local dwelling. Fluorescence-labeled CMMCs were detected in all vessel layers 4 weeks after delivery. Colonies of cells that localized to the lumen and stained for endothelial markers were also identified. Local CMMC administration at the time of balloon injury accelerated reendothelialization at 4 weeks compared with saline (P<0.05). Moreover, CMMC delivery markedly improved endothelium-dependent vasoreactivity at 4 weeks compared with saline (P<0.005). Finally, CMMC treatment reduced neointimal formation by 55% at 4 weeks (P<0.05). CONCLUSIONS: These data demonstrate that delivery of CMMCs to balloon-injured arteries is associated with accelerated reendothelialization, enhanced endothelium-dependent vasoreactivity, and reduced neointimal formation. Thus, delivery of autologous CMMCs represents a novel vasculoprotective approach to attenuate the response to acute vascular injury.

Caveolin-1 can regulate vascular smooth muscle cell fate by switching platelet-derived growth factor signaling from a proliferative to an apoptotic pathway.

BACKGROUND: Caveolin-1 is a regulator of signaling events originating from plasma membrane microdomains termed caveolae. This study was performed to determine the regulatory role of caveolin-1 on the proliferative events induced by platelet-derived growth factor (PDGF) in vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: Treatment of VSMCs with PDGF for 24 hours resulted in a loss of caveolin-1 protein expression and plasma membrane-associated caveolae, despite a 3-fold increase in caveolin-1 mRNA. Pretreatment of VSMCs with chloroquine, an inhibitor of lysosomal function, inhibited the PDGF-induced loss of caveolin-1. These studies demonstrated that caveolin-1 was a target of PDGF signaling events. Adenoviral overexpression of caveolin-1 was associated with a switch in PDGF-induced signaling events from a proliferative response to an apoptotic response. This overexpression inhibited PDGF-induced expression of cyclin D1 in the presence of unaffected mitogen-activated protein kinase activation. CONCLUSIONS: Taken together, these studies suggest that caveolin-1 is an inhibitor of PDGF proliferative responses and might be capable of transforming PDGF-induced proliferative signals into death signals.

Tissue factor pathway inhibitor deficiency enhances neointimal proliferation and formation in a murine model of vascular remodelling.

Tissue factor (TF) is a small-molecular-weight glycoprotein that initiates the extrinsic coagulation pathway but may have important noncoagulation vascular functions as well. Tissue factor pathway inhibitor (TFPI) is a major physiological inhibitor of TF-initiated coagulation. Enhancement of vascular TFPI either by overexpression using gene transfer or delivery of protein to the vessel has been shown to reduce neointimal formation. However, the inherent role of TFPI in this process has not been defined. To do so, we utilized a murine model of vascular remodeling using flow cessation in mice, which are heterozygous for a genetic deletion of the first Kunitz domain of TFPI or wild type littermates. The heterozygotic mice had 50% of wild type TFPI activity in plasma as well as vascular homogenates. To study the effect of TFPI deficiency on neointimal formation, age matched TFPI(K1)+/- and wildtype littermates underwent unilateral common carotid artery ligation. Mice were sacrificed at 4 weeks and the ligated carotid arteries were analyzed. There was a significantly greater neointima to media ratio and less luminal area in the TFPI(K1)+/- mice compared to their TFPI(K1)+/+ littermates. The proliferative index of intimal cells in TFPI(K1)+/- mice at 1 week was significantly higher compared to TFPI(K1)+/+ mice. We conclude that TFPI deficiency enhances neointimal formation and proliferation associated with flow cessation. This suggests that TFPI may regulate vascular remodeling primarily through modulation of neointimal formation.