Wulf D. Ito

Monocyte activation in angiogenesis and collateral growth in the rabbit hindlimb.

We have previously shown that monocytes adhere to the vascular wall during collateral vessel growth (arteriogenesis) and capillary sprouting (angiogenesis). In this study we investigated the association of monocyte accumulation with both the production of the cytokines-basic fibroblast growth factor (bFGF) and TNF-alpha-and vessel proliferation in the rabbit after femoral artery occlusion. In particular, we studied the effects of an increase in monocyte recruitment by LPS on capillary density as well as collateral and peripheral conductance after 7 d of occlusion. Monocytes accumulated around day 3 in collateral arteries when maximal proliferation was observed, and stained strongly for bFGF and TNF-alpha. In the lower limb where angiogenesis was shown to be predominant, macrophage accumulation was also closely associated with maximal proliferation (around day 7). LPS treatment significantly increased capillary density (424+/-26.1 n/mm2 vs. 312+/-20.7 n/mm2; P < 0.05) and peripheral conductance (109+/-33.8 ml/min/100 mmHg vs. 45+/-6.8 ml/min/100 mmHg; P < 0.05) as compared with untreated animals after 7 d of occlusion. These results indicate that monocyte activation plays a major role in angiogenesis and collateral artery growth.

Monocyte Chemotactic Protein-1 Increases Collateral and Peripheral Conductance After Femoral Artery Occlusion

Monocytes are activated during collateral artery growth in vivo, and monocyte chemotactic protein-1 (MCP-1) has been shown to be upregulated by shear stress in vitro. In order to investigate whether MCP-1 enhances collateral growth after femoral artery occlusion, 12 rabbits were randomly assigned to receive either MCP-1, PBS, or no local infusion via osmotic minipump. Seven days after occlusion, isolated hindlimbs were perfused with autologous blood at different pressures, measuring flows at maximal vasodilation via flow probe and radioactive microspheres, as well as peripheral pressures. This allowed the calculation of collateral (thigh) and peripheral (lower limb) conductances from pressure-flow tracings (slope of the curve). Collateral growth on postmortem angiograms was restricted to the thigh and was markedly enhanced with MCP-1 treatment. Both collateral and peripheral conductances were significantly elevated in animals with MCP-1 treatment compared with the control group, reaching values of nonoccluded hindlimbs after only 1 week of occlusion (collateral conductance, 70.6 +/- 19.23 versus 25.1 +/- 2.59 mL/min per 100 mm Hg; P < .01; peripheral conductance, 119.3 +/- 22.37 versus 45.4 +/- 6.80 mL/min per 100 mm Hg; P < .05). These results suggest that activation of monocytes plays an important role in collateral growth as well as in capillary sprouting.

Molecular Mechanisms of Coronary Collateral Vessel Growth

Growth of coronary collaterals can change markedly the natural history of coronary artery disease: Stenoses and occlusions of the coronary arteries, even of the left main coronary artery, can be survived without infarction provided that the stenosing process has not progressed too fast, since the process of collateral development by growth needs time (a few weeks).1 2 3 However, in most cases, thrombus formation proceeds faster than vascular growth and infarcts develop. In many cases, collaterals, although they cannot prevent infarction in the majority of cases, may limit the damage and infarcts are smaller than expected from the size of the region at risk.4 Understanding collateral growth may mean to be potentially and eventually able to stimulate it by the injection of drugs, by the injection of growth factors, or by somatic gene therapy in patients at risk of infarction. In the past we have shown that collaterals grow by DNA synthesis and mitosis of endothelial and smooth muscle cells.3 These cells are quiescent in normal adult arteries, with their population kinetics close to zero. Under abnormal conditions a rapid conversion to G1 can occur and the cell cycle can be completed in ≈22 hours.3 With rapidly progressing stenosis in dogs (3 days from the onset of stenosis to complete occlusion), the labeling index of the endothelium of the midzone segment reached 7.5% and was followed by a wave of smooth muscle cell mitosis of only slightly lesser magnitude. Since controlled and regulated growth does not proceed without the presence and action of growth factor peptides and their receptors, several groups have investigated that aspect.5 6 7 8 The growth factors that are potentially involved in the process of cardiac collateralization are aFGF, bFGF, VEGF, IGF-1, and PDGF.7 9 10 11 12 13 …

Ultrastructure and molecular histology of rabbit hind-limb collateral artery growth (arteriogenesis)

Abstract Previous studies in the canine heart had shown that the growth of collateral arteries occurs via proliferative enlargement of pre-existing arteriolar connections (arteriogenesis). In the present study, we investigated the ultrastructure and molecular histology of growing and remodeling collateral arteries that develop after femoral artery occlusion in rabbits as a function of time from 2 h to 240 days after occlusion. Pre-existent arteriolar collaterals had a diameter of about 50 µm. They consisted of one to two layers of smooth muscle cells (SMCs) and were morphologically indistinguishable from normal arterioles. The stages of arteriogenesis consisted of arteriolar thinning, followed by transformation of SMCs from the contractile- into the proliferative- and synthetic phenotype. Endothelial cells (ECs) and SMCs proliferated, and SMCs migrated and formed a neo-intima. Intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) showed early upregulation in ECs, which was accompanied by accumulation of blood-derived macrophages. Mitosis of ECs and SMCs started about 24 h after occlusion, whereas adhesion molecule expression and monocyte adhesion occurred as early as 12 h after occlusion, suggesting a role of monocytes in vascular cell proliferation. Treatment of rabbits with the pro-inflammatory cytokine MCP-1 increased monocyte adhesion and accelerated vascular remodeling. In vitro shear-stress experiments in cultured ECs revealed an increased phosphorylation of the focal contacts after 30 min and induction of ICAM-1 and VCAM-1 expression between 2 h and 6 h after shear onset, suggesting that shear stress may be the initiating event. We conclude that the process of arteriogenesis, which leads to the positive remodeling of an arteriole into an artery up to 12 times its original size, can be modified by modulators of inflammation.

Tissue Resident Cells Play a Dominant Role in Arteriogenesis and Concomitant Macrophage Accumulation

Collateral growth is characterized by macrophage accumulation, suggesting an important role of circulating cells. To study origin and function of macrophages during arteriogenesis, we related the extent of macrophage accumulation to vascular proliferation and investigated the fate of fluorescently (CMFDA) labeled blood cells that were injected at the time of femoral artery occlusion. The effect of bone marrow depletion via cyclophosphamide before femoral artery occlusion on collateral proliferation and macrophage accumulation was studied, and we looked for the presence of bone marrow—derived stem cells in the vicinity of growing collateral vessels. Finally, we investigated the arteriogenic effect of macrophage activation via MCP-1 in bone marrow—depleted animals. Maximal macrophage accumulation occurred during the first 3 days after femoral artery occlusion and paralleled the extent of vascular proliferation. Fluorescently labeled leukocytes homed to spleen and wound but they were absent in proliferating collateral arteries during maximal macrophage accumulation. Depletion of circulating cells did neither affect macrophage accumulation nor collateral growth. Staining of monocyte-depleted animals for BrdUrd and ED2, &agr;SMA, or VE-Cadherin demonstrated local proliferation of macrophages and vascular cells, whereas C-Kit, SSEA1, or Thy1-positive bone marrow–derived stem cells were not detectable. Enhancement of macrophage accumulation via MCP-1 was independent of circulating monocytes and promoted arteriogenesis in the absence of direct effects on vascular cells. We propose that the initial phase of vascular growth is characterized by local proliferation of tissue resident precursors rather than by migration of blood born cells. The full text of this article is available online at http://circres.ahajournals.org.

Carcinoembryonic antigen–related cell adhesion molecule 1 modulates vascular remodeling in vitro and in vivo

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a cellular adhesion molecule of the Ig superfamily, is associated with early stages of angiogenesis. In vitro, CEACAM1 regulates proliferation, migration, and differentiation of murine endothelial cells. To prove that CEACAM1 is functionally involved in the regulation of vascular remodeling in vivo, we analyzed 2 different genetic models: in Ceacam1-/- mice, the Ceacam1 gene was deleted systemically, and in CEACAM1(endo+) mice, CEACAM1 was overexpressed under the control of the endothelial cell-specific promoter of the Tie2 receptor tyrosine kinase. In Matrigel plug assays, Ceacam1-/- mice failed to establish new capillaries whereas in CEACAM1(endo+) mice the implants were vascularized extensively. After induction of hind limb ischemia by femoral artery ligation, Ceacam1-/- mice showed significantly reduced growth of arterioles and collateral blood flow compared with their WT littermates. In agreement with a causal role of CEACAM1 in vascular remodeling, CEACAM1(endo+) mice exhibited an increase in revascularization and collateral blood flow after arterial occlusion. Our findings indicate that CEACAM1 expression is important for the establishment of newly formed vessels in vivo. Hence CEACAM1 could be a future target for therapeutic manipulation of angiogenesis in disease.

Coronary artery anomalies Part II: recent insights from clinical investigations.

Angeborene Anomalien der Koronargefäße finden sich in 0,2–1,2% der Bevölkerung. Sie verursachen 12% der mit sportlicher Aktivität assoziierten plötzlichen Todesfälle und 1,2% der nicht im Rahmen sportlicher Aktivitäten auftretenden plötzlichen Todesfälle. Wir bieten eine Übersicht über die Fortschritte, die hinsichtlich der embryologischen Grundlagen dieser Koronaranomalien erzielt wurden und diskutieren neuere Erkenntnisse zu ihrer Diagnostik und Therapie. Der hier vorgelegte zweite Teil unserer Übersicht stellt aktuelle Konzepte zur Definition von koronarer Anomalität dar und informiert über Inzidenz, Verlauf und Prognose einzelner Entitäten. Besonderes Augenmerk richten wir auf die Diskussion von Möglichkeiten zum nicht-invasiven Screening größerer Bevölkerungskollektive und zur invasiven Diagnostik für die Risikostratifikation bei bereits diagnostizierter Koronaranomalie. Hierbei beleuchten wir besonders die klinische Problematik von Anomalien wie dem Abgang einer Koronararterie aus dem gegenüberliegenden Koronarsinus (ACAOS), Koronarfisteln und Myokardbrücken, die nur bei einer kleinen Gruppe von Patienten tatsächlich zu Komplikationen führen. Schließlich stellen wir die aktuellen Empfehlungen zur Diagnostik und Therapie von Koronaranomalien dar. Congenital anomalies of the coronary arteries occur in 0.2–1.2% of the general population; they cause 12% of sports-related sudden cardiac deaths and 1.2% of non-sports-related deaths. We review some of the substantial advances that have been made both, in the understanding of the embryonic development of the coronary arteries and in the clinical diagnosis and management of their anomalies. In this second part of our review we elucidate recent approaches to defining coronary anomalies and provide information on their incidence and prognosis. In addition, we discuss the options for screening large populations for potentially lethal coronary malformations and elucidate the role of invasive diagnostic modalities such as intravascular ultrasound, flow wire and pressure wire. The clinical relevance of coronary anomalies is discussed particularly for the ill-defined group of anomalies that only occasionally cause severe clinical events comprising anomalous origination of a coronary artery from the opposite sinus (ACAOS), coronary artery fistulae and myocardial bridging. Finally, we provide an update on current diagnostic and therapeutic recommendations.

Coronary artery anomalies. Part I: Recent insights from molecular embryology.

Angeborene Anomalien der Koronargefäße finden sich in 0,2–1,2% der Bevölkerung und können mit erheblicher Morbidität und Mortalität assoziiert sein. Diese Arbeit liefert eine Übersicht zum aktuellen Stand aus Sicht der Embryologie (Teil I) und zur klinischen Diagnostik und Therapie (Teil II). Im vorliegenden ersten Teil der Arbeit bieten wir eine Übersicht zur koronaren Vaskulogenese, Angiogenese und embryonalen Arteriogenese. Hierbei beleuchten wir besonders die Rolle von Vorläuferzellen wie beispielsweise der epikardialen Vorläuferzellen, der kardialen Neuralleistenzellen und Vorläuferzellen des peripheren Reizleitungssystems. Darüber hinaus stellen wir die Rolle verschiedener Wachstumsfaktoren (beispielsweise FGV, HIF 1, PDGF B, TGFβ1, VEGF und VEGFR-2) und Gene (beispielsweise FOG-2, VCAM-1, Bves und RALDH2) bei der Regulation einzelner Schritte der koronaren Gefäßbildung dar. Dieser Teil der Übersicht möchte die Vielzahl von Möglichkeiten und Mechanismen zur Entstehung koronarer Anomalitäten verdeutlichen. Deshalb verweisen wir besonders auf Ergebnisse aus Experimenten, die eine systematische Beziehung definierter Störungen auf molekularer Ebene mit koronarer Anomalie erkennen lassen. Besonders gehen wir hierbei auf die Rolle der Neuralleiste bei der Entwicklung von Koronaranomalien und deren Assoziation mit Anomalien der Aortenwurzel und Aortenklappe ein. Congenital anomalies of the coronary arteries occur in 0.2–1.2% of the general population and may cause substantial cardiovascular morbidity and mortality. We review some of the advances that have been made both, in the understanding of the embryonic development of the coronary arteries (part I) and in the clinical diagnosis and management of their anomalies (part II). In this first part of our review we elucidate basic mechanisms of coronary vasculogenesis, angiogenesis and embryonic arteriogenesis. Moreover, we review the role of cellular progenitors such as epicardium-derived cells, cardiac neural crest cells and cells of the peripheral conduction system. Then we discuss the role of growths factors (such as FGV, HIF 1, PDGF B, TGFβ1, VEGF, and VEGFR-2) and genes (such as FOG-2, VCAM-1, Bves, and RALDH2) at different states of coronary development. and we discuss the role of the cardiac neural crest in the concurrence of coronary anomalies with aortic root malformations. This part of the article is designed to review major determinants of coronary vascular development to provide a better understanding of the multiplicity of options and mechanisms that may give rise to coronary anomaly. To this end, we highlight results from experiments that provide insight in mechanisms of coronary malformation

Proteome Analysis of Migrating versus Nonmigrating Rat Heart Endothelial Cells Reveals Distinct Expression Patterns

Migration of endothelial cells plays an important role during angiogenesis and the late remodeling phase of arteriogenesis. To investigate mechanisms responsible for cell migration, the authors subcloned a rat heart endothelial cell line (RHE) into a migrating and a nonmigrating cell line (RHE-A and RHE-neg, respectively). Both cell lines form cobblestone patterns in confluent cultures similar to the originating cell line, but RHE-neg cells grow in dense cell islets of several layers whereas RHE-A cells grow in a less dense monolayer. Both cell lines show the same expression pattern of known endothelial cell surface antigens (e.g., FIK-1). The authors used two-dimensional gel electrophoresis technique to look for differentially regulated proteins with possible functional importance for cell migration. The analysis of the cytosolic fraction as well as the membrane fraction revealed differences in the protein expression patterns of RHE-neg and RHE-A cells. Regulated spots were isolated and analyzed by mass spectrometry (MS/MS technique), leading to the identification of proteins potentially responsible for endothelial cell migration, e.g., the intermediate filament vimentin that was exclusively expressed in RHE-A cells. The authors thus have generated a reproducible model that allows the analysis of the proteome responsible for endothelial cell migration.

Tissue macrophages: "satellite cells" for growing collateral vessels? A hypothesis.

It has been demonstrated in several studies that collateral growth is associated with accumulation of macrophages around proliferating vessel. Macrophages are known to secrete vascular growth factors and metalloproteinases. Both are necessary for the development of a proper vasculature. Recent studies suggest that certain subpopulations of macrophages are also capable of transdifferentiating into vascular cells. There are good reasons to assume that shear force rises dramatically in preexisting arteriolar shunts after occlusion of the main supplying vessel. Based upon these two findings it was hypothesized that high shear forces lead to homing of circulating monocytes to the growing collateral artery. The majority of studies, however, indicate that monocytes home under low shear force conditions. Our own observations in monocyte depleted animals suggest that proliferation and transdifferentiation of tissue macrophages occurs locally in growing collateral vessels and is independent of circulating cells. We thus propose that local proliferation and transdifferentiation of tissue macrophages rather than homing of circulating monocytes play a major role in arteriogenesis.