Formation Of New Capillaries Research Articles

Effect of Electrical Stimulation on Arteriogenesis and Angiogenesis after Bilateral Femoral Artery Excision in the Rabbit Hind-Limb Ischemia Model

The effects of electrical stimulation (ES) on arteriogenesis (the opening of preexisting collaterals) and angiogenesis (formation of new capillaries) were studied after acute bilateral hind limb ischemia was induced via bilateral femoral artery excision in a rabbit model. The study evaluated the rabbit hind limbs' normal response to acute ischemia and to application of ES by calculating changes in arterial and capillary densities. Comparisons were made with our prior study, in which the femoral artery was unilaterally excised, as we attempted to expand on the topics of arteriogenesis and angiogenesis. Twelve adult New Zealand white rabbits were randomly assigned to 1 of 2 series. In Series 1, the control group, both femoral arteries were excised and no ES was applied. In Series 2, both femoral arteries were excised and ES was applied to the left limb. One lead was implanted into the left adductor muscle near the site of the excised left femoral artery (Series 2), and a stimulator (Thera, Medtronic, Inc, Minneapolis, MN) was implanted in a separate pocket. ES was applied at a rate of 3 V, 30 contractions per minute, beginning immediately after surgery and continuously for 1 month. Angiography was performed in all 12 rabbits 1 month after surgery to establish the anatomy of the collateral vessels and to demonstrate that the femoral artery stump continued to be an end artery. Contrast-opacified arteries (COAs) that crossed the grid's midline, and the total number of grid lines intersected by COAs, were tallied according to an established method. Capillary density was calculated as the number of capillaries per square millimeter of muscle. In Series 1, after 1 month, the number of COAs crossing the grid's midline was 4.5 +/-1.5 on the left and 4.8 +/-1.2 on the right side. In Series 2, the number of COAs crossing the grid's midline was 7.9 +/-1.8 on the left side (p<0.05 vs Series 1) and 5.9 +/-1.6 on the right side of the same rabbit (p=NS vs Series 1). In Series 1, 36.7 +/-5.4 and 30.5 +/-7.7 total intersections were crossed by COAs on the left and right sides, respectively. In Series 2, total grid intersections crossed by COAs were 48.4 +/-8.5 and 47.5 +/-9.1 in the left and right sides, respectively (p<0.001 vs series 1). Baseline capillary density before femoral artery excision was 180.2 +/-21.3/mm(2). The capillary densities on the left sides were 94.2 +/-19.1 and 264.5 +/-7.6 in Series 1 and 2, respectively (p<0.001). The right sides showed a similar pattern with capillary densities of 88.5 +/-37.2 and 135.8 +/-6.8 (p<0.05) in Series 1 and 2, respectively. When capillary density was compared on the left and right sides of the same rabbit in Series 2, a statistically significant increase was also found; 264.5 +/-7.6 vs 135.8 +/-6.8 (p<0.001) in the left and right sides, respectively. Comparisons of the effect of electrical stimulation and the body's normal physiologic response to acute ischemia revealed a significant increase in the opening of preexisting collaterals (arteriogenesis) and the promotion of capillary density (angiogenesis) with the use of electrical stimulation.

Beneficial Effects of Circulating Progenitor Endothelial Cells Activated by Angiotensin Receptor Antagonists

The formation of new capillaries to provide oxygen supply for ischemic tissues or tumors was believed to be exclusively mediated by the proliferation and migration of existing endothelial cells. However, increasing evidence suggests that circulating cells home to sites of ischemia and contribute to adult neovascularization. It is likely that these processes are of importance in the long-term evolution of vascular adaptation of patients with ischemic diseases and possibly of hypertensive patients. Actually, ischemia is a major component of most of end-target damages of hypertension; thus, treatments and/or nonpharmacological therapy (such as physical exercise training for example) aiming to promote neovascularization could have new and unexpected beneficial effects. Bahlmann et al report, in this issue of Hypertension , that treatment with angiotensin II receptor antagonists increases the number of regenerative endothelial progenitor cells (EPCs) in patients with type II diabetes.1 In 1997, Asahara et al reported that CD34-positive mononuclear cells in the human peripheral blood incorporated into the foci of vascular injury and differentiated into vascular endothelial cells.2 Labeled human CD34-positive mononuclear cells isolated from peripheral blood and administered to nude mice with ischemic hindlimb were able to be incorporated into the ischemic muscle tissue and differentiated into endothelial cells. Further studies from the same group demonstrated that a certain population of bone marrow cells, now identified as EPCs, is recruited in the foci of neovascular formation and …

Growth factor signal transduction defects in the cardiovascular system

Growth factors are important molecules mediating both the development as well as adaptive and pathological changes within the cardiovascular system. Growth factors therefore mediate both beneficial and nonbeneficial effects. The beneficial actions include the improvement of endothelial function, stimulation of vascular repair, the formation of new capillaries (angiogenesis), and the growth of collateral arteries (arteriogenesis). These actions represent the conceptual basis for the therapeutic use of growth factors, based on the idea to therapeutically induce or accelerate a given beneficial process. The beneficial effects of growth factors have to be separated from effects where growth factors act as mediators of pathological processes such as atherogenesis and plaque destabilization. This review article focuses on the physiological and beneficial effects of growth factors in the vessel wall and describes and explains the dysfunction of these effects under certain conditions now referred to as "growth factor signal transduction defects".

Effects of retinoic acid steroidal analogs on human leukemic HL60 cell proliferation in vitro and on angiogenesis in vivo

Retinoic acid (RA) can be regarded as a pharmacological agent commonly used for its ability to affect growth and differentiation of a variety of cell types, such as acute promyelocytic leukemic and endothelial cells. In the present work we studied the effect of all-trans-RA (ATRA) and its steroidal analogs EA-4, EA-136 and EA-137 on the growth of human promyelocytic HL-60 cells in vitro. The specific steroidal substrates were chosen in order to further investigate their ability to improve the pharmacological properties of conjugated antileukemic agents. ATRA decreased the number of HL60 cells from the first 24 h after its addition to the cell culture medium. The decrease was significant at concentrations higher than 10(-5) M. All the analogs tested also decreased the number of HL60 cells with an IC50 similar to that of ATRA, except for EA-4 whose IC50 was almost two orders of magnitude lower than that of ATRA, 72 h after its addition to the cell culture medium. Since angiogenesis is important for the growth of hematological malignancies, we furthermore studied the effect of ATRA and its analogs on the formation of new capillaries in the in vivo chicken embryo chorioallantoic membrane (CAM). ATRA, EA-136 and EA-137 induced angiogenesis in the CAM, increased the layer of CAM keratinocytes, and resulted in a significant degree of extravasation. EA-4 had no effect on either angiogenesis or tissue structure in general. It seems that the retinoid EA-4 is a promising agent for the inhibition of human leukemia cell growth.

Role of Inflammatory Mediators in Angiogenesis

The angiogenic process involves several cell types and mediators, which interact to establish a specific microenvironment suitable for the formation of new capillaries from pre-existing vessels. Angiogenesis occurs in several physiological and pathological conditions, such as embryo development and wound healing, diabetic retinopathy and tumours. Inflammatory cells, namely monocytes/macrophages, T lymphocytes and neutrophils, fully participate in the angiogenic process by secreting cytokines that may affect endothelial cell (EC) functions, including EC proliferation, migration and activation. Angiogenesis is the result of a net balance between the activities exerted by positive and negative regulators. With regards to inflammatory cells and endothelium cross-talk, such balance is conceptually very similar to that of pro-inflammatory and anti-inflammatory mediators that modulate an appropriate inflammatory response. In this review we will mainly discuss the relevance of both physiological and pathological inflammatory processes in angiogenesis, with particular regards to microenvironmental contribution. We will also describe some of the most relevant pro-inflammatory cytokines in the modulation of the angiogenic process. Furthermore, we will concentrate on what has been recently reported about the mechanism by which some of these cytokines are induced during inflammation to promote a suitable microenvironment for angiogenesis and tumour progression. Pro-angiogenic cytokines, such as IL-1 and TNF, and anti-angiogenic cytokines such as IFN-gamma and IL-12, will be briefly described. We will try to provide a rationale for the use of both cytokines and cytokine blockades as novel potential pharmaceutical targets to modulate angiogenesis in chronic inflammation as well as in cancer.

Physical Modelling and Simulations of Tumour Growth and Angiogenesis: Predictions and New Hypotheses

The initial stages of tumour growth (avascular phase) are characterised by a low nutrient availability, which soon become a limiting factor for the progression of the neoplasm. Normally a transition to a vascular phase occurs, during which cancer cells stimulate the proliferation of endothelial cells belonging to vessels, hence the formation of new capillaries. The newly formed vascular system rapidly approaches the tumour surface and even infiltrates it, providing additional nutrients which allow further growth (angiogenesis). Blocking the process, might induce tumour to latency, with the consequent implications from therapeutical point of view. In the present contribution we will consider angiogenesis as a case study to show how mathematical models help in the interpretation and quantification of the experimental results.

Senescence and its bypass in the vascular endothelium

Vascular endothelial cells line the interior of blood vessels. As in other cell types, the proliferative lifespan of endothelial cells is limited; after a given number of replication cycles, they undergo senescence. Angiogenesis, the formation of new capillaries from pre-existing vasculature, is a process that involves endothelial cell proliferation. Angiogenesis thus has the possibility to be limited by the occurrence of senescence in the endothelial cell population. While there is evidence that endothelial cells undergo senescence in vivo, there are also data implying that endothelial senescence can be delayed or prevented in certain situations. Such a prevention of senescence would allow continued endothelial cell proliferation and continued angiogenesis in both physiological and pathological settings. This review discusses endothelial cell senescence and its bypass in vitro and in vivo.

Up-regulation of 94-kDa glucose-regulated protein by hypoxia-inducible factor-1 in human endothelial cells in response to hypoxia

Hypoxic environment in solid tumor is known to favor cell survival and to initiate the formation of new capillaries. In this work, we identified by 2D gel analysis 94-kDa glucose-regulated protein (GRP94) as being upregulated in human endothelial cells in response to hypoxia. Three putative hypoxia responsive elements (HRE) were found in the GRP94 promoter. Competition experiments of HIF-1 DNA binding using specific probes containing each HRE sequence of the GRP94 promoter clearly evidenced that HIF-1 binds these sequences with high affinity. The human GRP94 promoter was then cloned upstream of the luciferase gene and showed enhanced activity in hypoxic conditions. Mutation of two of the three HREs present in this promoter completely inhibited the hypoxia-induced increase in luciferase activity.

Distal angiogenesis: a new concept for lung vascular morphogenesis.

Although several molecular players have been described that play a role during the early phases of lung development, it is still unknown how the vasculature develops in relation to the airways. Two opposing models describe development of lung vasculature: one suggests that both vasculogenesis and angiogenesis are involved, whereas the second describes vasculogenesis as the primary mechanism. Therefore, we examined the development of the murine pulmonary vasculature through a morphological analysis from the onset of lung development [9.5 days postcoital (dpc)] until the pseudoglandular stage (13.5 dpc). We analyzed fetal lungs of Tie2-LacZ transgenic mice as well as serial sections of wild-type lungs stained with endothelial-specific antibodies (Flk-1, Fli-1, and PECAM-1). Embryos were processed with intact blood circulation to maintain the integrity of the vasculature; hence individual vessels could be identified with accuracy through serial section analysis. Furthermore, circulating primitive erythrocytes, formed exclusively by the blood islands in the yolk sac, are trapped in vessels during fixation, which proves the connection with the embryonic circulation. We report that from the first morphological sign of lung development, a clear vascular network exists that is in contact with the embryonic circulation. We propose distal angiogenesis as a new concept for early pulmonary vascular morphogenesis. In this model, capillary networks surround the terminal buds and expand by formation of new capillaries from preexisting vessels as the lung bud grows. The fact that at an early embryonic stage a complete vascular network exists may be important for the general understanding of embryonic development.

188 Targeted delivery of endostatin using an antibody-endostatin fusion protein resulted in enhanced inhibition of tumor growth

Endostar, a novel recombinant human endostatin expressed and purified in Escherichia coli with an additional nine-amino acid sequence and forming another his-tag structure, was approved by the SFDA in 2005 for the treatment of non-small-cell lung cancer. But its mechanism of action has not been illustrated before. In this study, we examined the antiangiogenic activities of endostar in vitro and in vivo. The results showed that endostar suppressed the VEGF-stimulated proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Endostar blocked microvessel sprouting from rat aortic rings in vitro. Moreover, it could inhibit the formation of new capillaries from pre-existing vessels in the chicken chorioallantoic membrane (CAM) assay and affect the growth of vessels in tumor. We further found the antiangiogenic effects of endostar were correlated with the VEGF-triggered signaling. Endostar suppressed the VEGF-induced tyrosine phosphorylation of KDR/Flk-1(VEGFR-2) as well as the overall VEGFR-2 expression and the activation of ERK, p38 MAPK, and AKT in HUVECs. Collectively, these data indicated the relationship between endostar and VEGF signal pathways and provided a molecular basis for the antiangiogenic effects of endostar.

190 Adaphostin (NSC680410) down regulates VEGF expression in human umbilical vein endothelial cells under hypoxia

Endostar, a novel recombinant human endostatin expressed and purified in Escherichia coli with an additional nine-amino acid sequence and forming another his-tag structure, was approved by the SFDA in 2005 for the treatment of non-small-cell lung cancer. But its mechanism of action has not been illustrated before. In this study, we examined the antiangiogenic activities of endostar in vitro and in vivo. The results showed that endostar suppressed the VEGF-stimulated proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Endostar blocked microvessel sprouting from rat aortic rings in vitro. Moreover, it could inhibit the formation of new capillaries from pre-existing vessels in the chicken chorioallantoic membrane (CAM) assay and affect the growth of vessels in tumor. We further found the antiangiogenic effects of endostar were correlated with the VEGF-triggered signaling. Endostar suppressed the VEGF-induced tyrosine phosphorylation of KDR/Flk-1(VEGFR-2) as well as the overall VEGFR-2 expression and the activation of ERK, p38 MAPK, and AKT in HUVECs. Collectively, these data indicated the relationship between endostar and VEGF signal pathways and provided a molecular basis for the antiangiogenic effects of endostar.

967. Angiotensinogen Inhibits Growth and Angiogenesis of Human Mammary Carcinoma and Prevents Pulmonary Metastasis of Murine Melanoma Cancer

It is now well established that a solid tumor cannot grow beyond a volume of a few mm3 without the formation of new capillaries to supply tumor cells with nutrients and oxygen, and to allow removal of waste products. We have already shown that recombinant human angiotensinogen (AGT) can inhibit endothelial cell proliferation and migration, capillary-like tube formation, and neovascularisation of the chick chorioallantoic membrane1. In order to determine whether these anti-angiogenic activities could impact tumor growth, we have delivered AGT by gene transfer into mice bearing experimental tumors using a replication defective adenovirus (AdAGT).

Characterization and biological activities of recombinant human plasminogen kringle 1–3 produced in Escherichia coli

Angiogenesis, the formation of new capillaries from preexisting blood vessels, is involved in many pathological conditions, for example, tumorigenesis, diabetic retinopathy, and rheumatoid arthritis. Angiostatin, which contains the kringle 1–4 domains of plasminogen, is known to be a potent inhibitor of angiogenesis and a strong suppressor of various solid tumors. In this study, we expressed recombinant protein containing the kringle 1–3 domains of human plasminogen in Escherichia coli and investigated its biological activities. The protein was successfully refolded from inclusion bodies and purified at a 30% overall yield, as a single peak by HPLC. The purified recombinant protein had biochemical properties that were similar to those of the native form, which included molecular size, lysine-binding capacity, and immunoreactivity with a specific antibody. The recombinant protein was also found to strongly inhibit the proliferation of bovine capillary endothelial cells in vitro, and the formation of new capillaries on chick embryos. In addition, it suppressed the growth of primary Lewis lung carcinoma and B16 melanoma in an in vivo mouse model. Our findings suggest that the recombinant kringle 1–3 domains in a prokaryote expression system have anti-angiogenic activities, which may be useful in clinical and basic research in the field of angiogenesis.

Antiangiogenic therapy in hematologic malignancies.

Angiogenesis is defined as the formation of new capillaries from prexisting blood vessels and plays an important role in the progression of solid tumors and hematologic malignancies. Markers of angiogenesis correlate with clinical characteristics in leukemia and non-Hodgkin's-lymphoma, serving as predictors of poor prognosis. Antiangiogenic effects of chemotherapeutics as well as of novel drugs such as farnesyltransferase inhibitors and tyrosine kinase inhibitors such as Gleevec might contribute to their therapeutic potential. Thalidomide which has antiangiogenic effects and direct cytotoxic effects was found to be effective in multiple myeloma and is considered as an established treatment modality for patients with refractory or relapsed multiple myeloma. Thalidomide has a significant therapeutic effect in myelodysplastic syndrome (MDS) by improving cytopenia and achieving independence of transfusion therapy in a subset of patients. Preliminary data indicate activity of specific VEGF receptor tyrosine kinase (RTK) inhibitors in multiple myeloma (MM) and acute myeloid leukemia (AML). The positive correlation between increased levels of angiogenic cytokines and clinical response to VEGF-RTK inhibitors and thalidomide indicates the relevance of detecting angiogenesis markers to identify best candidate patients for specific approaches. Including antiangiogenic drugs into treatment protocols for hematologic malignancies is an important task for future clinical studies.

Endothelial progenitor cells: more than an inflammatory response?

The formation of new capillaries (angiogenesis) may be of clinical importance in facilitating reperfusion and regeneration of hibernating cardiac tissue after myocardial infarction and in microvascular ischemia. Evidence is accumulating that as part of the response to hypoxia, bone marrow-derived circulating endothelial progenitor cells (CEPs) are mobilized and subsequently differentiate into proper endothelial cells. There are also indications that such CEPs can facilitate endothelial repair and angiogenesis in vivo. It is not clear yet, however, whether these CEPs are essential for these adaptive processes or what the relative contribution of CEP is compared with that of other mononuclear inflammatory cells that are mobilized to areas of ischemia. Moreover, there are still many uncertainties about how cardiovascular risk factors alter CEP function. Particularly when therapeutically mobilizing CEPs, a further understanding of this issue is essential to assess the risk of potentially harmful side effects of altered CEP function.

Molecular basis of angiogenesis and cancer.

Angiogenesis is a term that describes the formation of new capillaries from a pre-existing vasculature. This process is very important in physiologic conditions because it helps healing injured tissues, and in female populations it helps forming the placenta after fertilization and reconstructs the inside layer of the uterus after menstruation. Angiogenesis is the result of an intricate balance between proangiogenic and antiangiogenic factors and is now very well recognized as a powerful control point in tumor development. In this particular environment, the fine modulation among proangiogenic and antiangiogenic factors is disrupted, leading to inappropriate vessels growth. In this review, we discuss the molecular basis of angiogenesis during tumor growth and we also illustrate some of the molecules that are involved in this angiogenic switch.

Regulation of angiogenesis by Th1- and Th2-type cytokines.

Angiogenesis is a complex process, where several cell types and mediators interact to establish a specific microenvironment suitable for the formation of new capillaries from pre-existing vessels. Such biological processes occur in several physiological conditions, such as embryo development and wound healing, as well as in pathological conditions, including tumours and diabetic retinopathy. T lymphocytes, neutrophils and monocytes fully participate in the angiogenic process by secreting cytokines that may control endothelial cell (EC) proliferation, their survival and apoptosis, as well as their migration and activation. Angiogenesis is the result of a net balance between the activities exerted by positive and negative regulators. This balance is conceptually very similar to that of the Th1/Th2 cells that modulate an appropriate and specific immune response. Th1 or Th2 cytokines may control angiogenesis directly, by acting on cell growth and differentiation, indirectly by inducing the release of other cytokines in the microenvironment, and by modulating the expression of specific receptors, involved in the control of angiogenic processes, such as EC proliferation and migration. In this review we will mainly discuss the role of Th1- and Th2-type cytokines in the angiogenic process, emphasizing the complexity of the cytokine and leukocyte/EC network, and highlighting the care that needs to be taken when designing new therapeutic interventions involving Th1 and Th2 cytokines.

Tissular insemination of progenitor endothelial cells: the problem, and a suggested solution.

The contribution of circulating precursor endothelial cells (CPEC) to adult angiogenesis is now well established. However, the mechanism of their tissular engrafting remains poorly understood. The classical paradigm of "sprouting" cannot accommodate the main features of the CPEC-based angiogenic process. Additionally, vasculogenesis based on the differentiation of angioblasts, as defined in the embryonic stages, is not applicable to adult neo-vascularization either. In search for a solution to this dilemma, I suggest that the ability of monocytes/macrophages to produce tunnels, as effect of their protease-dependent migration in the extracellular matrices, is instrumental for the tissular insemination of CPEC. Here I present in vivo and in vitro experimental evidence for the existence of tunnels, and for their colonization by monocytes/macrophages and by other cells, including CPEC. As a paradigm of CPEC behavior, the tunneling model (in an extended sense) may also explain the propagation of the endothelium with arteriolar phenotype within the pre-existent downstream capillary network. Thus, the sprouting mechanism might be a valid explanation for the formation of new capillaries and venules, whereas CPEC would contribute mostly, if not exclusively, to the extension of arteriolar branches of microvasculature. Adult angiogenesis occurs therefore as a multifunctional process based on intercellular cooperation, in which there are involved endothelial cells (EC) or their precursors, as well as other cell types. In specific circumstances, the lumen (i.e. the tunnel) may occur before the "definitive" microvessel. Therefore the very notion of microvessel may need to be extended, to include the tunnels.

Hu protein R-mediated posttranscriptional regulation of VEGF expression in rat gastrocnemius muscle.

Hypoxic exercise increases VEGF expression and the formation of new capillaries. In addition to hypoxia-inducible factor regulation at the transcriptional level, VEGF message stabilization is also a key regulatory step for VEGF expression. In vitro experiments have identified Hu protein R (HuR) as a potential posttranscriptional regulator of VEGF gene expression. Here, we report that in rat skeletal muscle (gastrocnemius muscle), 1) HuR binds to a known regulatory sequence located in the VEGF mRNA 3'-untranslated region (1,631-1,678 bp); 2) HuR specifically binds to the A/U-rich element AUUUUA (1,665-1,670 bp) and an additional A/U-rich region containing the consensus sequence UUUUUUA (1,658-1,664 bp); 3) binding of HuR to VEGF mRNA is seen already 5 min after acute ischemia, remaining elevated throughout a 60-min ischemic period; 4) a second inducible HuR-VEGF mRNA binding factor is evident 30 and 60 min postischemia; and 5) VEGF mRNA and protein levels are increased 20 and 30 min, respectively, after acute ischemia. These findings suggest that acute ischemia induces a rapid binding of HuR to the VEGF mRNA 3'-untranslated region. In skeletal muscle, this specific protein-RNA interaction may be an important posttranscriptional regulatory mechanism for increasing VEGF expression in response to hypoxia or acute ischemia.

Neuropilin-1 Participates in Wound Angiogenesis

Angiogenesis, the formation of new capillaries from existing vasculature, plays an essential role in tissue repair. The rapid onset and predominance of proangiogenic factors optimizes healing in damaged tissues. One factor that directly mediates wound vessel angiogenesis is vascular endothelial growth factor (VEGF). Although much is known about the biology of VEGF and its cognate receptors, VEGFR1 and VEGFR2, the role of a recently identified co-receptor for VEGF, neuropilin-1, is not well understood. Using a murine model of dermal wound repair, we found that neuropilin-1 was abundantly expressed on new vasculature in healing wounds. Moreover, mice treated with anti-neuropilin-1 antibodies exhibited a significant decrease in vascular density within these wounds (67% decrease, P = 0.0132). In in vitro assays, VEGF induced formation of endothelial cord-like structures on collagen gel and endothelial cell migration toward VEGF was inhibited by antibodies directed against neuropilin-1. These results provide both in vitro and in vivo evidence for a critical role of neuropilin-1 in wound angiogenesis.