Formation Of Capillary Vessels Research Articles

Enhanced Angiogenic Efficacy through Controlled and Sustained Delivery of FGF-2 and G-CSF from Fibrin Hydrogels Containing Ionic-Albumin Microspheres

Neo-vessel formation in ischemic tissues relies on numerous growth factors and cell fractions for the formation of mature, stable, functional vasculature. However, the efforts to regenerate tissues typically rely on the administration of a single growth factor or cells alone. Conversely, polymeric matrices have been investigated extensively to deliver multiple growth factors at pre-determined rates to form stable blood vessels in ischemic tissues. We report on a novel sequential delivery system of a fibrin hydrogel containing ionic-albumin microspheres that allows for the controlled release of two growth factors. The use of this system was investigated in the context of therapeutic angiogenesis. Material properties were determined based on degree of swelling measurements and degradation characteristics. Release kinetics of model angiogenic polypeptides FGF-2 and G-CSF were determined using ELISA and the bioactivity of released protein was evaluated in human endothelial cell cultures. The release of growth factors from ionic-albumin microspheres was significantly delayed compared to the growth factor released from fibrin matrices in the absence of spheres. The scaffolds were implanted in a murine critical limb ischemia model at two concentrations, 40 ng (low) and 400 ng (high), restoring 92% of the blood flow in a normally perfused limb using a fibrin hydrogel releasing FGF-2 containing albumin–PLL microspheres releasing G-CSF (measured by LDPI at the high concentration), a 3.2-fold increase compared to untreated limbs. The extent of neo-vessel formation was delineated by immunohistochemical staining for capillary density (CD-31+) and mature vessel formation (α-SMA+). In conclusion, our study demonstrated that the release kinetics from our scaffold have distinct kinetics previously unpublished and the delivery of these factors resulted in hindlimb reperfusion, and robust capillary and mature vessel formation after 8 weeks compared to either growth factor alone or bolus administration of growth factor.

Prevention and Recovery of Hibernating Myocardium by Microvascular Repair

> “From small things, big things one day come.” > > —Bruce Springsteen Coronary artery disease, ischemic cardiomyopathy, and congestive heart failure continue to confer significant morbidity and mortality despite advances in medical therapy, percutaneous coronary interventions, and surgical revascularization.1,2 This unmet clinical need is the rationale for novel therapeutic strategies like therapeutic angiogenesis. Unlike traditional revascularization, which focuses on macrovascular solutions, angiogenesis seeks to improve the microcirculation by stimulating new capillary and collateral arterial vessel formation. Recently, there has been significant discussion on traditional revascularization, in particular the benefit of revascularization with coronary artery bypass surgery (CABG) in patients with left ventricular (LV) dysfunction in the Surgical Treatment for Ischemic Heart Failure (STICH) Trial.3 As opposed to mechanical revascularization, whether by CABG or percutaneous coronary intervention, therapeutic angiogenesis has the advantage of improving coronary microcirculation by harnessing existing physiological processes to address a biological problem. Article see p 1059 A variety of strategies to promote therapeutic angiogenesis have been investigated, including delivery of proangiogenic genes, growth factors, and autologous stem cells aspirated or mobilized from the bone marrow or peripheral blood. These various approaches have demonstrated promising results in the treatment of organ-specific manifestations of systemic atherosclerosis, including peripheral artery disease, refractory angina, and ischemic cardiomyopathy, particularly in animal models and early human clinical trials of cell-based therapies. However, some of the larger therapeutic angiogenesis clinical trials using individual growth factors have not corroborated the exciting early results, indicating that additional work is necessary to determine the ideal agent(s) and delivery mechanism(s) to maximize efficacy and durability of the intervention while limiting off-target effects of angiogenesis. Additionally, thoughtful trial design and selection of end points that reflect the physiology of therapeutic angiogenesis and improvement in the coronary microcirculation such as measurement of coronary flow reserve will be critical …

Heat Shock Cognate Protein 70 Is Essential for Akt Signaling in Endothelial Function

Heat shock protein 70s (Hsp70s) are molecular chaperones that protect cells from damage in response to various stress stimuli. However, the functions and mechanisms in endothelial cells (ECs) have not been examined. Herein, we investigate the role of Hsp70s, including heat shock cognate protein 70 (Hsc70), which is constitutively expressed in nonstressed cells (ie, ECs). The Hsp70 inhibitor, KNK437, significantly decreased vascular endothelial growth factor (VEGF)-induced cell migration and tube formation in vitro. KNK437 inhibited the phosphorylation of VEGF-induced Akt and endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells. In a mouse hind limb model of vascular insufficiency, intramuscular inhibition of Hsp70s attenuated collateral and capillary vessel formation. Silencing the Hsc70 gene by short interfering RNA abolished VEGF-induced Akt phosphorylation and VEGF-stimulated human umbilical vein endothelial cell migration and tube formation. As the molecular mechanisms, Hsc70 knockdown reduced the expression of phosphatidylinositol 3-kinase. Collectively, Hsc70 plays a significant role in ECs via the phosphatidylinositol 3-kinase/Akt pathway. Hsc70 may provide the basis for the development of new therapeutic strategies for angiogenesis.

Novel Roles of GATA1 in Regulation of Angiogenic Factor AGGF1 and Endothelial Cell Function

AGGF1 is an angiogenic factor, and its deregulation is associated with a vascular malformation consistent with Klippel-Trenaunay syndrome (KTS). This study defines the molecular mechanism for transcriptional regulation of AGGF1 expression. Transcription of AGGF1 starts at two nearby sites, -367 and -364 bp upstream of the translation start site. Analyses of 5'- and 3'-serial promoter deletions defined the core promoter/regulatory elements, including two repressor sites (from -1971 to -3990 and from -7521 to -8391, respectively) and two activator sites (a GATA1 consensus binding site from -295 to -300 and a second activator site from -129 to -159). Both the GATA1 site and the second activator site are essential for AGGF1 expression. A similar expression profile was found for GATA1 and AGGF1 in cells (including various endothelial cells) and tissues. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays demonstrated that GATA1 was able to bind to the AGGF1 DNA in vitro and in vivo. Overexpression of GATA1 increased expression of AGGF1. We identified one rare polymorphism -294C>T in a sporadic KTS patient, which is located in the GATA1 site, disrupts binding of GATA1 to DNA, and abolishes the GATA1 stimulatory effect on transcription of AGGF1. Knockdown of GATA1 expression by siRNA reduced expression of AGGF1, and resulted in endothelial cell apoptosis and inhibition of endothelial capillary vessel formation and cell migration, which was rescued by purified recombinant human AGGF1 protein. These results demonstrate that GATA1 regulates expression of AGGF1 and reveal a novel role for GATA1 in endothelial cell biology and angiogenesis.

Delivery of Basic Fibroblast Growth Factor from Gelatin Microsphere Scaffold for the Growth of Human Umbilical Vein Endothelial Cells

One of the major obstacles for engineering large tissue or organs such as the liver in vitro is the insufficient supply of nutrients and oxygen to the cells growing inside the scaffold, which reduces cell viability significantly. Therefore, vascularization of the scaffolding system is necessary for successful engineering of such tissues. In this study, we investigated the use of gelatin microsphere as scaffold to culture human umbilical vein endothelial cells, which is considered to be the basis and premise for the formation of blood vessels. The gelatin microspheres were crosslinked with different concentrations of glutaraldehyde to study the effects of crosslinking extent on the growth of endothelial cells. The swelling ratios of the gelatin microspheres decreased from 5.9 +/- 0.8 to 3.9 +/- 0.6 with the increase of the crosslinking extent. Basic fibroblast growth factors (bFGFs), which can improve endothelial cell proliferation as well as stimulate the formation of capillary vessels, were incorporated into the gelatin microspheres through ionic complexation. Sustained delivery of the growth factors was achieved for at least 2 weeks. The proliferation of the cells cultured on the bFGF-encapsulated microspheres was improved by about two times as compared to control and about 1.3 times as compared to blank microspheres, which indicated that the bioactivity of bFGF was well maintained, and the delivery of the growth factors directly to the cells significantly improved the success of this tissue engineering system.

Signaling via the Tgf-β type I receptor Alk5 in heart development

Trophic factors secreted both from the endocardium and epicardium regulate appropriate growth of the myocardium during cardiac development. Epicardially-derived cells play also a key role in development of the coronary vasculature. This process involves transformation of epithelial (epicardial) cells to mesenchymal cells (EMT). Similarly, a subset of endocardial cells undergoes EMT to form the mesenchyme of endocardial cushions, which function as primordia for developing valves and septa. While it has been suggested that transforming growth factor-βs (Tgf-β) play an important role in induction of EMT in the avian epi- and endocardium, the function of Tgf-βs in corresponding mammalian tissues is still poorly understood. In this study, we have ablated the Tgf-β type I receptor Alk5 in endo-, myo- and epicardial lineages using the Tie2-Cre, Nkx2.5-Cre, and Gata5-Cre driver lines, respectively. We show that while Alk5-mediated signaling does not play a major role in the myocardium during mouse cardiac development, it is critically important in the endocardium for induction of EMT both in vitro and in vivo. Moreover, loss of epicardial Alk5-mediated signaling leads to disruption of cell–cell interactions between the epicardium and myocardium resulting in a thinned myocardium. Furthermore, epicardial cells lacking Alk5 fail to undergo Tgf-β-induced EMT in vitro. Late term mutant embryos lacking epicardial Alk5 display defective formation of a smooth muscle cell layer around coronary arteries, and aberrant formation of capillary vessels in the myocardium suggesting that Alk5 is controlling vascular homeostasis during cardiogenesis. To conclude, Tgf-β signaling via Alk5 is not required in myocardial cells during mammalian cardiac development, but plays an irreplaceable cell-autonomous role regulating cellular communication, differentiation and proliferation in endocardial and epicardial cells.

A hybrid protein comprising ATF domain of pro-UK and VAS, an angiogenesis inhibitor, is a potent candidate for targeted cancer therapy

Directional and controllable degradation of extracellular matrix mediated by the uPA-uPA receptor (uPAR) system is ubiquitously implicated in tumor establishment, invasion and metastasis. Targeting the excessive activation of this system as well as the proliferation of the tumor vascular endothelial cell would be expected to prevent tumor neovasculature and halt the tumor development. In this study, we created a fusion protein (ALV), comprising the aminoterminal fragment (ATF) of urokinase and VAS, the antiangiogenic functional domain of vasostatin. The antitumor activity of this hybrid molecule was evaluated with both in vitro and in vivo experiments. Cell adhesion and motility assays demonstrated that ALV, owing to its ATF moiety, could interact with uPAR on the tumor cell surface with high affinity and specificity, and thereby might competitively inhibit the plasmin activation by localized urokinase and contribute to the suppression of tumor invasion. These results and speculations were validated by zymography assay and Matrigel invasion assay. In addition, ALV exhibited an improved inhibitory efficacy against endothelial cell (EC) proliferation and capillary vessel formation in a 3D angiogenesis model, proving that ATF and VAS, when fused into a chimeric molecule, cooperatively inhibited angiogenesis by targeting both the interaction of uPA and uPAR on cell surface (by ATF) and EC proliferation (mainly by VAS). Animal model confirmed that, at the same molar dose, ALV produced significantly higher therapeutic benefit than VAS and ATF in terms of tumor growth delay and mice survival prolongation. Conclusively coupling VAS with the uPAR ligand ATF resulted in an improved antineoplastic activity, which may show a novel avenue for the design of tumor therapeutic drugs.

Growth Factor Therapy in Atherosclerotic Disease-Friend or Foe

Stimulation of neovascularization (angiogenesis, arteriogenesis) has emerged as a promising new strategy to treat patients with coronary disease. These strategies aim to improve cardiac function by ensuring myocardial perfusion and to reduce the risk of myocardial infarction. While angiogenesis describes a de-novo formation of small caliber capillary vessels, arteriogenesis leads to the outgrowth of pre-existing arterioles into large conductance collateral arteries. Inflammatory cells (e.g. monocytes), which can produce and secrete growth factors and cytokines, mediate both processes. Several trials have shown that intra-coronary infusion of growth factors or progenitor cells can improve left ventricular function after arterial occlusion. Despite these encouraging results, potential unfavorable effects on plaque progression and stability should not be neglected. Destabilization of atherosclerotic plaques leads to plaque rupture, intravascular thrombosis and tissue infarction. Increased neovascularization of the plaque (e.g. by angiogenesis) is thought to arise from the adventitial vasa vasorum, leading to an abnormal vascular development. This network of immature vessels is a viable source of invading inflammatory cells that can contribute to plaque instability. Furthermore, intra-plaque hemorrhages can lead to accumulation of erythrocyte membranes in the plaque that are rich in phospholipids and free cholesterol, promoting lesion instability through necrotic core expansion. Future angiogenic and arteriogenic approaches need to take these pitfalls into account and should focus on stimulation of vessel growth in combination with neutral or even beneficial effects on plaque formation and composition. This review discusses the delicate balance between the benefits and the drawbacks of therapeutic strategies to influence angiogenesis and arteriogenesis.

The Vascular Compartments of Neovascularization: Spotlight on the Microcirculation

In this review, the compartments of the vasculature will be discussed with respect to their potential contribution to the build-up of a differentiated and functionally meaningful vessel system. Chronic ischemia of muscle tissue results in microvessel rarification, which represents a potential therapeutic target, given the viability of the parenchyma. In particular, a chain of microcirculatory events will be described which--after specific endothelial activation by growth factors--enhances capillary and microcirculatory vessel formation, Backward signalling is introduced as potential mechanism to induce collateral growth, recruiting pre-existent macrovessel networks for blood supply to the increased microcirculatory cross sectional area. Potential signal cascades will be discussed.

MATHEMATICAL MODELING OF TUMOR-INDUCED ANGIOGENESIS

Angiogenesis, the growth of a network of blood vessels, is a crucial component of solid tumor growth, linking the relatively harmless avascular and the potentially fatal vascular growth phases of the tumor. As a process, angiogenesis is a well-orchestrated sequence of events involving endothelial cell migration and proliferation; degradation of tissue; new capillary vessel formation; loop formation (anastomosis) and, crucially, blood flow through the network. Once there is flow associated with the nascent network, subsequent growth evolves both temporally and spatially in response to the combined effects of angiogenic factors, migratory cues via the extracellular matrix, and perfusion-related hemodynamic forces in a manner that may be described as both adaptive and dynamic. In this article, we first present a review of previous theoretical and computational models of angiogenesis and then indicate how recent developments in flow models are providing insight into antiangiogenic and chemotherapeutic drug treatment of solid tumors.

Toxicity results and early outcome data on a randomized phase II study of docetaxel ± bevacizumab for locally advanced, unresectable breast cancer

3049 Background: Preclinical models of combination angiogenesis inhibitor bevacizumab (rhuMAbVEGF) and docetaxel demonstrate synergistic suppression of capillary vessel formation. Based upon these data, we developed a randomized phase II trial in order to evaluate the vascular effects on tumor regression with combination bevacizumab/docetaxel vs. docetaxel in the treatment of locally advanced breast cancer. Methods: 49 patients (pts) were randomized to receive neoadjuvant therapy with bevacizumab (10 mg/kg qowk) and docetaxel (two 8-week cycles of 35 mg/m2 weekly x 6 with a 2 wk break) (BD=24) or docetaxel (D=25) alone. Eligible pts had locally unresectable breast cancer with (n=6) or without distant metastasis (n=43); 16 patients presented with inflammatory breast cancer. Pts whose disease responded, sequentially underwent definitive surgery (4 weeks after BD or D), radiation, 4 cycles of conventional Adriamycin/cyclophosphamide, and tamoxifen or anastrazole (if ER/PR+). Results: Among the 49 pts: 7 clinical CRs, 32 PRs, 5 NR, and 5 PD. Of the 37 pts who underwent surgery: the median number of pathologically positive lymph nodes (LN) was 1 (BD=6, D=1; p=0.228); range 0–20; 43% were LN negative. Neoadjuvant treatment toxicity for both arms was acceptable with no significant differences between the two arms. Grade 4 toxicity included BD - new papillary thyroid cancer (1), neutropenia (1), hyperuricemia (1) and colon perforation (1); and D: - hyperglycemia (1) and hyperuricemia (1). 21 patients in each arm experienced a grade 3 toxicity. There were no episodes of uncontrolled hypertension, proteinuria, or thrombosis. Delayed wound healing (unable to start radiation w/in 6 weeks of surgery) occurred in 8 pts: BD=5; D=3 (p=0.691). Only 1 pt (D) experienced a change in LVEF by > 15% or below the institution’s lower limit of normal. Conclusions: Neoadjuvant therapy for locally advanced breast cancer using docetaxel with bevacizumab is well tolerated. Further studies are required to determine the added efficacy from bevacizumab. Correlative studies on impact of treatment on angiogenesis will be reported separately. (Sponsored by grants: K23CA 87725–01, M01 RR 00080, UO1 CA 62502, 5P30 CA43703-NCI/AVON, Aventis) No significant financial relationships to disclose.

Mathematical modelling of dynamic adaptive tumour-induced angiogenesis: Clinical implications and therapeutic targeting strategies

Angiogenesis, the growth of a network of blood vessels, is a crucial component of solid tumour growth, linking the relatively harmless avascular growth phase and the potentially fatal vascular growth phase. As a process, angiogenesis is a well-orchestrated sequence of events involving endothelial cell migration, proliferation; degradation of tissue; new capillary vessel (sprout) formation; loop formation (anastomosis) and, crucially, blood flow through the network. Once there is blood flow associated with the nascent network, the subsequent growth of the network evolves both temporally and spatially in response to the combined effects of angiogenic factors, migratory cues via the extracellular matrix and perfusion-related haemodynamic forces in a manner that may be described as both adaptive and dynamic. In this paper we present a mathematical model which simultaneously couples vessel growth with blood flow through the vessels— dynamic adaptive tumour-induced angiogenesis (DATIA). This new mathematical model presents a theoretical and computational investigation of the process and highlights a number of important new targets for therapeutic intervention. In contrast to earlier flow models, where the effects of perfusion (blood flow) were essentially evaluated a posteriori, i.e. after generating a hollow network, blood flow in the model described in this paper has a direct impact during capillary growth, with radial adaptations and network remodelling occurring as immediate consequences of primary anastomoses. Capillary network architectures resulting from the dynamically adaptive model are found to differ radically from those obtained using earlier models. The DATIA model is used to examine the effects of changing various physical and biological model parameters on the developing vascular architecture and the delivery of chemotherapeutic drugs to the tumour. Subsequent simulations of chemotherapeutic treatments under different parameter regimes lead to the identification of a number of new therapeutic targets for tumour management.

Relationship between bleomycin-induced pulmonary fibrosis and vascular endothelial cell injury

To explore the relationship between the injury of vascular endothelial cells and formation of lung fibrosis by bleomycin (BLM) in rats. The rats of experimental groups were treated with bleomycin intratracheally to induce pulmonary fibrosis. The expression of vascular endothelial growth factor (VEGF) in pulmonary tissues were analyzed qualitatively and quantitatively by immunohistochemistry and image analysis system. (1) HISTOLOGY: Edema in rat alveoli and alveolar septum, inflammatory cells exudation, degeneration and necrosis of type I and type II alveolar epithelial cells (AETI and AETII), ruptured alveolar basement membrane, as well as swollen vascular endothelial cells and karyopyknosis were observed in 3 d and 7 d after treatment with BLM. AETII proliferation, with more fibroblasts in alveolar septum, and new capillary vessel formation in 7, 14 d, as well as thickened alveolar septum, damaged alveolar structure, and obvious pulmonary tissue fibrosis in 28 d after treatment with BLM were observed. (2) Immunohistochemistry: in normal control, VEGF expressed weakly in pulmonary tissue distributing mainly in AETII, bronchial epithelial cells, alveolar macrophages and leydig's cells. While in bleomycin treated groups, the expression of VEGF increased markedly. The expression in AETII, and pulmonary macrophage were significantly higher than that in control in 3 d to 28 d (P < 0.05, P < 0.01). The rat leydig's cells also had higher expression of VEGF in 7, 14, 28 d (P < 0.05, P < 0.01). The high expression of VEGF is related to vascular endothelial cells injury which may be one of important factors in the formation of bleomycin-induced pulmonary fibrosis.

Phase II trial of neoadjuvant docetaxel with or without bevacizumab in patients with locally advanced breast cance

727 Background: The anti-angiogenic agent, bevacizumab (rhuMAbVEGF) is a humanized monoclonal antibody against VEGF which, when combined with docetaxel in preclinical models, results in synergistic suppression of capillary vessel formation. Based on these data, a randomized phase II trial was developed to evaluate the vascular effects on tumor regression with combination bevacizumab/docetaxel vs. docetaxel in the treatment of locally advanced breast cancer. Methods: 33 patients (pts) were randomized to receive neoadjuvant therapy with bevacizumab (10 mg/kg qowk) and docetaxel (two 8-week cycles of 35 mg/m2 weekly x 6 with a 2 wk break) or docetaxel alone. Eligible pts had locally unresectable breast cancer with or without metastasis. Pts who responded underwent definitive surgery, radiation, 4 cycles of adjuvant conventional Adriamycin/cyclophosphamide, followed by tamoxifen (if ER/PR+). Results: 26 pts completed pre-operative treatment. 3 pts with inflammatory breast cancer were not operable at the conclusion of therapy because of residual disease. The median number of pathologically + lymph nodes (ln) was 1 (range 0–16); 40% were ln negative. The median size of residual tumor was 3.2cm (range 0–14cm). Toxicity included: grade 4 neutropenia-4; grade 3: anorexia-3; GI bleed-1; stomatitis-3; neuropathy-1; wound healing-1. There was no change in LVEF, no hypertension, protienuria, or thrombosis. Preliminary analysis of correlative studies suggests a reduction in tumor Kep by DCE-MRI and a reduction of tumor microvessel density. Conclusions: Neoadjuvant therapy for locally advanced breast cancer using docetaxel with or without bevacizumab is well tolerated and effective. This study requires a total of 60 pts before an analysis can be made of a treatment difference in vascular and disease response. Laboratory correlatives evaluating effects on tumor vasculature are ongoing. (Sponsored by grants: K23CA 87725–01, M01 RR 00080, UO1 CA 62502, P30 CA43703S NCI/Avon) No significant financial relationships to disclose.

Phase II trial of neoadjuvant docetaxel with or without bevacizumab in patients with locally advanced breast cance

727 Background: The anti-angiogenic agent, bevacizumab (rhuMAbVEGF) is a humanized monoclonal antibody against VEGF which, when combined with docetaxel in preclinical models, results in synergistic suppression of capillary vessel formation. Based on these data, a randomized phase II trial was developed to evaluate the vascular effects on tumor regression with combination bevacizumab/docetaxel vs. docetaxel in the treatment of locally advanced breast cancer. Methods: 33 patients (pts) were randomized to receive neoadjuvant therapy with bevacizumab (10 mg/kg qowk) and docetaxel (two 8-week cycles of 35 mg/m2 weekly x 6 with a 2 wk break) or docetaxel alone. Eligible pts had locally unresectable breast cancer with or without metastasis. Pts who responded underwent definitive surgery, radiation, 4 cycles of adjuvant conventional Adriamycin/cyclophosphamide, followed by tamoxifen (if ER/PR+). Results: 26 pts completed pre-operative treatment. 3 pts with inflammatory breast cancer were not operable at the conclusion of therapy because of residual disease. The median number of pathologically + lymph nodes (ln) was 1 (range 0–16); 40% were ln negative. The median size of residual tumor was 3.2cm (range 0–14cm). Toxicity included: grade 4 neutropenia-4; grade 3: anorexia-3; GI bleed-1; stomatitis-3; neuropathy-1; wound healing-1. There was no change in LVEF, no hypertension, protienuria, or thrombosis. Preliminary analysis of correlative studies suggests a reduction in tumor Kep by DCE-MRI and a reduction of tumor microvessel density. Conclusions: Neoadjuvant therapy for locally advanced breast cancer using docetaxel with or without bevacizumab is well tolerated and effective. This study requires a total of 60 pts before an analysis can be made of a treatment difference in vascular and disease response. Laboratory correlatives evaluating effects on tumor vasculature are ongoing. (Sponsored by grants: K23CA 87725–01, M01 RR 00080, UO1 CA 62502, P30 CA43703S NCI/Avon) No significant financial relationships to disclose.

Myogenic Akt signaling regulates blood vessel recruitment during myofiber growth.

Blood vessel recruitment is an important feature of normal tissue growth. Here, we examined the role of Akt signaling in coordinating angiogenesis with skeletal muscle hypertrophy. Hypertrophy of C2C12 myotubes in response to insulin-like growth factor 1 or insulin and dexamethasone resulted in a marked increase in the secretion of vascular endothelial growth factor (VEGF). Myofiber hypertrophy and hypertrophy-associated VEGF synthesis were specifically inhibited by the transduction of a dominant-negative mutant of the Akt1 serine-threonine protein kinase. Conversely, transduction of constitutively active Akt1 increased myofiber size and led to a robust induction of VEGF protein production. Akt-mediated control of VEGF expression occurred at the level of transcription, and the hypoxia-inducible factor 1 regulatory element was dispensable for this regulation. The activation of Akt1 signaling in normal mouse gastrocnemius muscle was sufficient to promote myofiber hypertrophy, which was accompanied by an increase in circulating and tissue-resident VEGF levels and high capillary vessel densities at focal regions of high Akt transgene expression. In a rabbit hind limb model of vascular insufficiency, intramuscular activation of Akt1 signaling promoted collateral and capillary vessel formation and an accompanying increase in limb perfusion. These data suggest that myogenic Akt signaling controls both fiber hypertrophy and angiogenic growth factor synthesis, illustrating a mechanism through which blood vessel recruitment can be coupled to normal tissue growth.

Cyclooxygenase, lipoxygenase and tumor angiogenesis.

Arachidonic acid metabolism through cyclooxygenase (COX) and lipoxygenase (LOX) pathways generates various biologically active lipids that play important roles in inflammation, thrombosis and tumor progression. Angiogenesis, the formation of new capillary vessels from preexisting ones, underpins a number of physiological processes and participates in the development of several pathological conditions such as arthritis, cancer and various eye diseases. The formation of new capillary vessels is a multistep process that involves endothelial cell proliferation, migration and tube formation. In the present review, we survey the literature on the regulation of angiogenesis by arachidonate metabolites, especially those from the COX and 12-LOX pathways in the context of tumor growth, and put forward some unanswered but important questions for future studies.

Regeneration of gastric mucosa during ulcer healing is triggered by growth factors and signal transduction pathways

An ulcer is a deep necrotic lesion penetrating through the entire thickness of the gastrointestinal mucosa and muscularis mucosae. Ulcer healing is a complex and tightly regulated process of filling the mucosal defect with proliferating and migrating epithelial and connective tissue cells. This process includes the re-establishment of the continuous surface epithelial layer, glandular epithelial structures, microvessels and connective tissue within the scar. Epithelial cells in the mucosa of the ulcer margin proliferate and migrate onto the granulation tissue to re-epithelialize the ulcer. Growth factors, such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), trefoil peptides (TP), platelet derived growth factor (PDGF) and other cytokines produced locally by regenerating cells, control re-epithelialization and the reconstruction of glandular structures. These growth factors, most notably EGF, trigger epithelial cell proliferation via signal transduction pathways involving EGF-R- MAP (Erk1/Erk2) kinases. Granulation tissue, which develops at the ulcer base, consists of fibroblasts, macrophages and proliferating endothelial cells, which form microvessels under the control of angiogenic growth factors. These growth factors [bFGF, vascular endothelial growth factor (VEGF) and angiopoietins] promote angiogenesis—capillary vessel formation—thereby allowing for the reconstruction of microvasculature in the mucosal scar, which is essential for delivery of oxygen and nutrients to the healing site. The primary trigger to activate expression of angiogenic growth factors and their receptors appears to be hypoxia. During ulcer healing expression of growth factor genes is tightly regulated in a temporally and spatially ordered manner.

Indomethacin Inhibits Endothelial Cell Proliferation by Suppressing Cell Cycle Proteins and PRB Phosphorylation: A Key to Its Antiangiogenic Action?

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit angiogenesis in vivo and in vitro, but the mechanism of this action is unclear. Angiogenesis—formation of new capillary vessels—requires endothelial proliferation, migration, and tube formation. It is stimulated by basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The cell cycle is regulated positively by cyclins and negatively by cyclin-dependent kinase inhibitors (CKI) and the retinoblastoma protein (pRb). Since the effects of NSAIDs on cell cycle-regulatory proteins in endothelial cells remain unknown, we examined the effect of indomethacin on bFGF-stimulated endothelial cell proliferation and on cell cycle regulatory proteins in rat primary aortic endothelial cells (RAEC). Indomethacin significantly inhibited basal and bFGF-stimulated endothelial cell proliferation. This inhibition correlated significantly with reduced cyclin D1 and increased p21 protein expression. Furthermore, indomethacin reduced pRb phosphorylation. These findings suggest that indomethacin arrests endothelial cell proliferation essential for angiogenesis by modulating cell cycle protein levels.

血管形成と再構築の基礎 血管新生の調節因子

Endothelial cells lining the lumen of vessels are maintained in the quiescent state and play important physiological roles. Yet, they can be de-differentiated and become one of the most rapidly proliferating of all cell types when stimulated. Angiogenesis or neovascularization is defined as the formation of new capillary vessels from existent microvessels, which plays a major role in the evolvement of a vascular supply in tissue during development or remodeling and disease. Angiogenesis is believed to be regulated by the balance between inducers and inhibitors. In this review article, I will summarize the molecules that regulate the process of angiogenesis.