Local Angiogenesis Research Articles

Heparanase mRNA expression during fracture repair in mice.

Bone fracture healing takes place through endochondral ossification where cartilaginous callus is replaced by bony callus. Vascular endothelial growth factor (VEGF) is a requisite for endochondral ossification, where blood vessel invasion of cartilaginous callus is crucial. Heparanase is an endoglucuronidase that degrades heparan sulfate proteoglycans (HSPG) and releases heparin-binding growth factors including VEGF as an active form. To investigate the role of heparanase in VEGF recruitment during fracture healing, the expression of heparanase mRNA and VEGF, and vessel formation were examined in mouse fractured bone. On days 5 and 7 after the fracture, when mesenchymal cells proliferated and differentiated into chondrocytes, heparanase mRNA was detected in osteo(chondro)clasts and their precursors, but not in the inflammatory phase (day 3). On day 10, both VEGF and HSPG were produced by hypertrophic chondrocytes of the cartilaginous callus and by osteoblasts of the bony callus; numerous osteo(chondro)clasts resorbing the cartilage expressed strong heparanase signals. Adjacent to the cartilage resorption sites, angiogenesis with CD31-positive endothelial cells and osteogenesis with osteonectin-positive osteoblasts were observed. On days 14 and 21, osteoclasts in the woven bone tissue expressed heparanase mRNA. These data suggest that by producing heparanase osteo(chondro)clasts contribute to the recruitment of the active form of VEGF. Thus osteo(chondro)clasts may promote local angiogenesis as well as callus resorption in endochondral ossification during fracture healing.

Cerebrum Repair with PHPMA Hydrogel Immobilized with Neurite-Promoting Peptides in Traumatic Brain Injury of Adult Rat Model

A poly[N-(2-hydroxypropyl) methacrylamide] (PHPMA) hydrogel immobilized with the neurite-promoting peptide sequence of xIKVAVx was synthesized and its structure was characterized. The PHPMA-IKVAV hydrogel displayed an interconnected porous structure. The ability of the hydrogel to support axonal outgrowth in the injured adult rats cerebrum cavity and to promote tissue regeneration was evaluated. After implantation for 4, 6, 12 and 18 weeks, the brain sections were processed for histological staining. The observations of the sections showed that the polymer hydrogel provided a structural, three-dimensional continuity across the defect and favoured reorganization of local wound-repair cells, angiogenesis and axonal growth into the hydrogel scaffold. Compared with the unmodified PHPMA hydrogel, the PHPMA-IKVAV hydrogel displayed greater ability to repair tissue defects in the cerebrum nervous system.

Cell-demanded release of VEGF from synthetic, biointeractive cell ingrowth matrices for vascularized tissue growth.

Local, controlled induction of angiogenesis remains a challenge that limits tissue engineering approaches to replace or restore diseased tissues. We present a new class of bioactive synthetic hydrogel matrices based on poly(ethylene glycol) (PEG) and synthetic peptides that exploits the activity of vascular endothelial growth factor (VEGF) alongside the base matrix functionality for cellular ingrowth, that is, induction of cell adhesion by pendant RGD-containing peptides and provision of cell-mediated remodeling by cross-linking matrix metalloproteinase substrate peptides. By using a Michael-type addition reaction, we incorporated variants of VEGF121 and VEGF165 covalently within the matrix, available for cells as they invade and locally remodel the material. The functionality of the matrix-conjugated VEGF was preserved and was critical for in vitro endothelial cell survival and migration within the matrix environment. Consistent with a scheme of locally restricted availability of VEGF, grafting of these VEGF-modified hydrogel matrices atop the chick chorioallontoic membrane evoked strong new blood vessel formation precisely at the area of graft-membrane contact. When implanted subcutaneously in rats, these VEGF-containing matrices were completely remodeled into native, vascularized tissue. This type of synthetic, biointeractive matrix with integrated angiogenic growth factor activity, presented and released only upon local cellular demand, could become highly useful in a number of clinical healing applications of local therapeutic angiogenesis.

Bioengineered implantation of megalin-expressing cells: a potential intracorporeal therapeutic model for uremic toxin protein clearance in renal failure.

Patients who have renal failure and are on dialysis therapy experience serious complications caused by low-molecular-weight uremic toxin proteins normally filtered by glomeruli and metabolized by proximal tubule cells (PTC). Dialysis-related amyloidosis is one such complication induced by systemic deposition of amyloid proteins derived from 12-kD beta(2)-microglobulin (beta(2)-m). Despite the use of high-flux membrane hemodialysis devices and direct absorbent columns, the removal of beta(2)-m is suboptimal, because the effects are transient and insufficient. Megalin is expressed in the apical membranes of PTC and recognized as a multiligand endocytic receptor that binds numerous low-molecular-weight proteins, including beta(2)-m. This study tested the feasibility of an intracorporeal therapeutic model of continuous beta(2)-m removal using megalin-expressing cell implantation. By cell association and degradation assays, rat yolk sac-derived L2 cells were identified to internalize and degrade beta(2)-m via megalin. The cells were effectively implanted within the subcutaneous tissues of nude mice using a type I collagen scaffold and a method inducing local angiogenesis. After nephrectomy and intraperitoneal injection with (125)I-beta(2)-m, it was found that the implanted cells took up the labeled ligand, efficiently removing it from the blood. Bioengineered implantation of megalin-expressing cells may represent a new supportive therapy for dialysis patients to compensate for the loss of renal protein metabolism and remove uremic toxin proteins.

New frontiers in cardiology: drug-eluting stents: Part II.

In this second part of the article, we will complete the review of drug-eluting stent technologies (Table 1) and discuss methodological and technical aspects of drug-eluting stents. View this table: TABLE 1. Drug-Eluting Stent Platforms Under Investigation ### Biological Agents (Continued) #### (1) Stents Eluting Antiproliferative Agents A number of antineoplastic medications have been considered for the prevention of restenosis. Paclitaxel and its derivatives have been the most investigated compounds of this group. ##### (a) Paclitaxel-Eluting Stents Paclitaxel (Taxol; Bristol-Myers Squibb) is a microtubule-stabilizing agent with potent antitumor activity.1 Many different platforms that use polymer coating or surface modifications to adhere paclitaxel onto the stents have been utilized over the past 2 years. ###### Preclinical Data Unlike other antimitotic agents, paclitaxel shifts the cytoskeleton equilibrium toward assembly, leading to reduced vascular cell proliferation, migration, and signal transduction.2 Paclitaxel is highly lipophilic, resulting in a rapid cellular uptake and a long-lasting effect in the cell.3 NIR stents (Boston Scientific Corp) coated with poly(lactide-co-Σ-caprolactone) copolymer and paclitaxel (200 μg/stent) were placed in porcine coronary arteries. Paclitaxel-eluting stents showed a marked reduction in neointimal and medial cell proliferation at all time points (7, 28, 56, and 180 days).4 However, arteries treated with paclitaxel showed incomplete healing, late persistence of a large number of macrophages, and fibrin deposition. Similar findings were observed with a stent platform coated with cross-linked biodegradable polymer (chondroitin sulfate and gelatin) and 42.0, 20.2, 8.6, or 1.5 μg of paclitaxel in rabbit iliac arteries.5 These studies indicate the need for a more controlled drug release of paclitaxel due to the narrow toxic-therapeutic window and high hydrophobic character of this compound. ###### Clinical Data: De Novo Lesions The QuaDS drug-eluting stent (Quanam Medical Corp) was the first drug-eluting stent implanted in human coronary arteries. This slotted tube stent has 50% of its surface area covered by multiple nonbiodegradable polyacrylate sleeves that release 7-hexanoyltaxol (called QP2 or taxane). Approximately 800 μg of the …

Intra-Arterial Bone Marrow Cell Transplantation Induces Angiogenesis in Rat Hindlimb Ischemia

Background: Bone marrow (BM) cells have been shown to augment local angiogenesis by differentiating vessels themselves and/or secreting paracrinally angiogenic growth factors. Herein, the angiogenic effects of intra-arterial BM mononuclear cell (BM-MNC) transplantation were evaluated in a rat ischemic hindlimb model. Methods: Unilateral hindlimb ischemia was created by excising the femoral artery and its branch in Lewis rats. BM-MNCs were isolated by centrifugation through a Histopaque density gradient. One week after excision of the unilateral femoral artery, BM-MNCs (5 × 10⁶ cells, Group A, n = 6) or PBS (Group B, n = 7) were injected into the ischemic thigh skeletal muscles at the six points with a gauge needle. Another injection of BM-MNCs (3 × 10⁷ cells, Group C, n = 6) or PBS (Group D, n = 7) was administered via the indwelling catheter in the right common iliac artery. Results: Four weeks after the BM-MNC transplantation, angiographic examination revealed the development of collateral vessels in both BM-MNC-transplanted groups. The difference in skin temperature between right and left hindlimbs was significantly reduced in both BM-MNC-transplanted groups (0.93 ± 0.15 vs. 2.84 ± 0.35 vs. 1.20 ± 0.26 vs. 2.61 ± 0.37°C, Group A vs. Group B vs. Group C vs. Group D, p < 0.05). Moreover, immunohistochemical analysis demonstrated that capillary endothelial cells were increased in both BM-MNC-transplanted groups. Conclusion: BM-MNC implantation was able to induce functional neovascularization in rat ischemic hindlimb. The intra-arterial administration offered similar levels of angiogenic activity as intramuscular injection.

Myocardial Angiogenesis Resulting in Functional Communications with the Left Cavity Induced by Intramyocardial High-Frequency Ablation: Histomorphology of Immediate and Long-Term Effects in Pigs

A previous study in rabbit hearts demonstrated the feasibility of creating transmyocardial channels by application of temperature-regulated high-frequency (HF) energy. Feasibility of creating non-transmural channels using a transvascular approach was tested in 14 pigs which were followed for 1 h, and 3 and 9 weeks (group A, B and C). Myocardial channels were found to be highly reproducible and patent in more than two thirds in group A. Channels were replaced by connective tissue during follow-up which contained newly formed small vessels. A functional communication between the left ventricular cavum and newly formed vessels of the channel remnants could be demonstrated in one heart of group C. Thus, intramyocardial HF ablation can be performed with high reproducibility to induce local angiogenesis.

Matrix Metalloproteinases-2 and-9 in Areas of Extracellular Matrix Destruction and Angiogenesis in Coronary Artery Aneurysms in Kawasaki Disease

Coronary artery aneurysms (CAA) can complicate Kawasaki Disease (KD) and lead to rupture, coronary thrombosis or coronary stenosis. CAA are characterized histologically by destruction of extracellular matrix (ECM), most notably elastic tissue in the vascular media; inflammatory cell infiltrates; and neovascularization. Matrix metaloproteinases (MMP) are known to regulate degradation and remodeling of the ECM and angiogenesis. MMP-2 and-9 have been implicated in the pathogenesis of abdominal aortic aneurysms and atherosclerosis, and elevated plasma levels of MMP-9 have been demonstrated in acute KD. We hypothesized that MMP are important in the pathogenesis of CAA development and vascular remodeling in KD. To determine if MMP are present in KD CAA, we performed immunohistochemistry for MMP-2 and -9 on paraffin-embedded formalin-fixed coronary artery tissue from 11 fatal acute KD cases and from 7 children who died of other causes. Although there was no significant quantitative difference in MMP-2 in KD and non-KD coronary arteries, there was a qualitative difference in the pattern of MMP-2 expression. In control and non-aneurysmal KD arteries, MMP-2 was present only in vascular smooth muscle cells and endothelial cells, whereas in KD CAA, MMP-2 was prominent in the thickened neointima, in smooth muscle cells migrating from the media into the neointima, and in endothelial cells in areas of adventitial angiogenesis. In contrast, there was a significant difference in the expression of MMP-9 in KD CAA and control coronary arteries (p≤0.01). MMP-9 was prominently expressed by mononuclear inflammatory cells in CAA but was not expressed in non-aneurysmal KD coronary arteries or in control subject coronary arteries. We conclude that MMP-2 and -9 are differentially expressed in CAA of KD patients when compared to non-aneurysmal KD or control coronary arteries. Differential expression of MMP may contribute to ECM degradation, neointimal proliferation, and local angiogenesis in KD CAA.

KLF5/BTEB2, a Krüppel-like zinc-finger type transcription factor, mediates both smooth muscle cell activation and cardiac hypertrophy.

Cardiac and vascular biology need to be approached interactively because they share many common biological features as seen in activation of the local renin-angiotensin system, angiogenesis, and extracellular matrix production. We previously reported KLF5/BTEB2, a Krüppel-like zinc-finger type transcription factor, to activate various gene promoters that are activated in phenotypically modulated smooth muscle cells, such as a nonmuscle type myosin heavy chain gene SMemb, plasminogen activator inhibitor-1 (PAI-1), iNOS, PDGF-A, Egr-1 and VEGF receptors at least in vitro. KLF5/BTEB2 mRNA levels are downregulated with vascular development but upregulated in neointima that is produced in response to vascular injury. Mitogenic stimulation activates KLF5/BTEB2 gene expression through MEK1 and Egr-1. Chromatin immunoprecipitation assay showed KLF5/BTEB2 to be induced and to bind the promoter of the PDGF-A gene in response to angiotensin II stimulation. In order to define the role of KLF5/BTEB2 in cardiovascular remodeling, we targeted the KLF5/BTEB2 gene in mice. Homozygous mice resulted in early embryonic lethality whereas heterozygous mice were apparently normal. However, in response to external stress, arteries of heterozygotes exhibited diminished levels of smooth muscle and adventitial cell activation. Furthermore, cardiac fibrosis and hypertrophy induced by continuous angiotensin II infusion. We also found that RARa binds KLF5/BTEB2, and that Am80, a potent synthetic RAR agonist, inhibits angiotensin II-induced cardiac hypertrophy. These results indicate that KLF5/BTEB2 is an essential transcription factor that causes not only smooth muscle phenotypic modulation but also cardiac hypertrophy and fibrosis.

Unique properties of 189 amino acid isoform of vascular endothelial growth factor in tumorigenesis

The 189 amino acid isoform of vascular endothelial growth factor (VEGF189) has been shown to be more strongly associated with the cell membrane than other isoforms of human VEGF (VEGF121, VEGF165). To analyze the biological activities of these VEGF isoforms on tumor growth, we transfected human VEGF121, VEGF165 or VEGF189 cDNA into the human colon cancer cell line SW-480, and established several clones overexpressing these VEGF isoforms. The total amounts of VEGF protein in the culture supernatants of the VEGF189-transfectants were less than those of VEGF121 and VEGF165-transfectants. These transfectants showed no significant differences in growth in culture. Nevertheless, the rate of in vivo tumor growth of VEGF189-transfectants was faster than or equivalent to that of VEGF121-transfectants, while the VEGF165-transfectant showed the greatest enhancement of tumor growth. The protein levels of VEGF were markedly increased only in the VEGF189-transfectants cultured in the presence of heparin. The enhanced in vivo tumor growth of VEGF189-transfectants can be partly explained by the cell-associated features of VEGF189 molecules. The VEGF189 molecule, which is strongly bound to the cell surface, has unique properties and high potential in local angiogenesis and tumor growth in the cancer inductive microenvironment.

Vascular Growth Factor Expression in a Rat Model of Severe Limb Ischemia

Background. In ischemic tissue hypoxia induces production of vascular growth factors, especially VEGF, which initiate local angiogenesis. Collateralization—or arteriogenesis—occurs at a distance from the ischemic tissue and depends on different growth factors such as FGF-2. A spatial discrepancy in endogenous growth factor production in limb ischemia may have implications for therapeutic angiogenesis. The present study elucidates if such spatial and temporal variation occurs. Materials and methods. A two-staged procedure was performed to generate severe long-lasting limb ischemia in 60 rats. At 1, 7, 28, and 56 days, subgroups were subjected to perfusion assessment with laser Doppler imaging and angiography. Muscle samples and foot skin were gathered to measure growth factor expression and signs of angiogenesis using immunohistochemistry. Results. There was an early twofold increase ( P < 0.05) in both VEGF and FGF-2 levels in distal muscle from the ischemic leg, but no significant rise in the thigh. The concentrations decreased over time with an exception for VEGF in soleus and FGF-2 in anterior tibial muscle, which remained high. An increased capillarity was noted ( P < 0.05) in soleus after 28 days, and the number of BrdU-positive ECs was elevated in all ischemic samples at 56 days. Collateral arteries were observed on the angiograms after 7 days. Conclusions. The results suggest that in limb ischemia any major increase in vascular growth factor production is limited to ischemic tissue. The spatial and temporal distribution patterns of growth factor production are complex and to a great extent influenced by inflammation.

Chemokine/chemokine receptor nomenclature.

Chemokine/chemokine receptor nomenclature.

Inflammation and cancer, the mastocytoma P815 tumor model revisited: Triggering of macrophage activation in vivo with pro‐tumorigenic consequences

Subcutaneous in vivo injections of cells of the mastocytoma line P815 in syngenic DBA/2 mice induce locally fast growing solid tumors. These have been used extensively as a cancer model to analyze and manipulate the relationship between tumor cells and host's immune defenses. We report that progression of P815 tumors in vivo was accompanied by a burst (Days 5-7) of local inflammatory cells recruitment and angiogenesis observed histologically, corroborated in vivo by MRI with gadolinium, overtranscription of macrophage activation marker genes, secretion of TNF-alpha by regional lymph node cells and concomitant systemic inflammation. No substantial overtranscriptions of either VEGF or IL-10 or TGF-beta genes were observed. Induction of COX-2 gene was a late event. To establish a possible relationship between the tumor-induced local, regional and systemic increase of pro-inflammatory mediators and progression of tumors in vivo, we carried out experiments deliberately modulating the inflammatory status of the recipient animals. Pretreatment of recipient animals by i.p. injection of thioglycolate accelerated P815 tumor growth. At the opposite, treatment of mice with either a COX-1 + COX-2 inhibitor (aspirin, 1 mg/day/mouse) or a specific COX-2 inhibitor (celecoxib, 0.13 mg/day/mouse) for 2 weeks after injection of tumor cells, significantly reduced the size and growth rate of tumors compared to control mice. Experiments carried out in vitro indicated that peritoneal macrophages from untreated animals were strongly activated by live P815 cells and by P815 membrane preparations. The tumor-induced inflammatory reaction could establish a local micro environment favoring tumor progression. The P815 tumor model might be helpful to recognize important factors controlling host/tumor relationship.

Coordinated Interaction of Neurogenesis and Angiogenesis in the Adult Songbird Brain

Neurogenesis proceeds throughout life in the higher vocal center (HVC) of the adult songbird neostriatum. Testosterone induces neuronal addition and endothelial division in HVC. We asked if testosterone-induced angiogenesis might contribute importantly to HVC neuronal recruitment. Testosterone upregulated both VEGF and its endothelial receptor, VEGF-R2/Quek1/KDR, in HVC. This yielded a burst in local HVC angiogenesis. FACS-isolated HVC endothelial cells produced BDNF in a testosterone-dependent manner. In vivo, HVC BDNF rose by the third week after testosterone, lagging by over a week the rise in VEGF and VEGF-R2. In situ hybridization revealed that much of this induced BDNF mRNA was endothelial. In vivo, both angiogenesis and neuronal addition to HVC were substantially diminished by inhibition of VEGF-R2 tyrosine kinase. These findings suggest a causal interaction between testosterone-induced angiogenesis and neurogenesis in the adult forebrain.

Overexpression of the p53-inducible brain-specific angiogenesis inhibitor 1 suppresses efficiently tumour angiogenesis.

The brain-specific angiogenesis inhibitor 1 gene has been isolated in an attempt to find fragments with p53 “functional” binding sites. As reported herein and by others, brain-specific angiogenesis inhibitor 1 expression is present in some normal tissues, but is reduced or lost in tumour tissues. Such data and its particular structure prompted the hypothesis that brain-specific angiogenesis inhibitor 1 may act as a mediator in the local angiogenesis balance. We herein demonstrate that brain-specific angiogenesis inhibitor 1 over-expression suppresses tumour angiogenesis, delaying significantly the human tumour growth in immunodeficient mice. The inhibitory effect of brain-specific angiogenesis inhibitor 1 was documented using our intravital microscopy system, strongly implicating brain-specific angiogenesis inhibitor 1 as a mediator in the control of tumour angiogenesis. In contrast, in vitro tumour cell proliferation was not inhibited by brain-specific angiogenesis inhibitor 1 transfection, whereas some level of cytotoxicity was assessed for endothelial cells. Immunohistochemical analysis of tumour samples confirmed a reduction in the microvessel density index in brain-specific angiogenesis inhibitor 1-overexpressing tumours. At messenger level, moderate changes could be detected, involving the down-regulation of vascular endothelial growth factor and collagenase-1 expression. Furthermore, brain-specific angiogenesis inhibitor 1 expression that was lost in a selection of human cancer cell lines could be restored by wild-type p53 adenoviral transfection. Brain-specific angiogenesis inhibitor 1 should be considered for gene therapy and development of efficient drugs based on endogenous antiangiogenic molecules.British Journal of Cancer (2002) 86, 490–496. DOI: 10.1038/sj/bjc/6600067 www.bjcancer.com© 2002 The Cancer Research Campaign

Cytokine-Dependent gp130 Receptor Subunit Regulates Rat Modified Axial-Pattern Epigastric Flap

We investigated axial-pattern flap survival by modulating the cytokine receptor component, gp130, to elucidate the mechanism of the cytokine receptor modulation subsequently leading to the initiation of local angiogenesis and blood-flow augmentation. Fifty-two male Wistar rats were divided into four groups. The flap size was 8 2 8 cm, with the medial middle branch of the superficial epigastric artery as the only nutrient supply vessel. Group 1 received only a control vehicle, group 2 received leukemia inhibitory factor(LIF), cDNA treated, group 3 received combined treatment of LIF and anti-gp130 antibody, and group 4 was the anti-gp130-treated group. All groups received 0.1 mL phosphate-buffered saline (PBS) via 30-gauge needle injected into the proximal site of the femoral artery after distal sites of the bifurcated femoral artery to epigastric artery and vein were clamped. All clamps were removed after 10 min of incubation of the injected agents. During the first 3 postoperative days, the flap survival was significantly more improved in the anti-gp130-treated and combined-treated groups than in the control (88 - 1.5% and 77 - 5.3 vs. 45 - 7.8, respectively, p < .01). Laser blood flowmetry on day 3 showed a significant increase of blood flow in three different flap sites in anti-gp130-treated and combined-treated groups than in the other groups ( p < .001). Microangiography showed remarkable differences between all four groups. The anti-gp130-treated flap showed augmented vessel formation at the base of the pedicle, which elongated the vessel interconnection up to the distal area. Strong gp130 transcripts and vascular endothelial growth factor (VEGF) expression were observed in all groups, as determined using reverse-transcription polymerase chain reaction (RT-PCR). The intensity of VEGF expression was the strongest in the anti-gp130-treated group. Attenuation of the gp130 receptor subunit can improve axial-pattern flap survival along with angiogenesis.

Quantification of vascular endothelial growth factor-A in leiomyomas and adjacent myometrium

Although uterine leiomyomas constitute the commonest benign tumour in women, the regulation of their growth is poorly understood. It is believed that angiogenesis, the process by which new capillaries develop from pre-existing blood vessels, may be involved. We therefore investigated the expression of vascular endothelial growth factor-A (VEGF-A), a primary regulator of angiogenesis, in leiomyoma tissue and the adjacent myometrium in 36 pre-menopausal women undergoing hysterectomy for leiomyomas, with or without prior treatment with gonadotrophin-releasing hormone analogue (GnRHa). In 5 microm sections prepared from archival paraffin-wax blocks, VEGF-A was demonstrated by standard immunohistochemistry using a monoclonal antibody. VEGF-A was expressed in 14 of 18 (77.8%) leiomyoma sections from women without GnRHa pretreatment, and in 15 of 18 (83%) of those from women with prior treatment. VEGF-A expression in the adjacent myometrium was much lower, being noted in two of 18 (11.1%) sections from women without prior GnRHa treatment and in one of 18 (5.5%) sections from tissue that had been subject to prior down-regulation. Moreover, when VEGF-A expression was present, expression was strong in leiomyomas (> or =20 focal areas/cm(2)), but not in adjacent myometrium. The differential expression of VEGF-A antigen in leiomyomas compared with the adjacent myometrium indicates that local angiogenesis may be important in the development and growth of these tumours. GnRHa therapy does not appear to alter this pattern of VEGF-A expression.

Angiopoietin-1, Unlike Angiopoietin-2, Is Incorporated into the Extracellular Matrix via Its Linker Peptide Region

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) affect angiogenesis differently during embryogenesis and tumorigenesis. In an attempt to understand the molecular basis underlying the distinct roles of those two homologous molecules, we investigated the association of Ang-1 and Ang-2 with the extracellular matrix (ECM). TA3 murine mammary carcinoma (TA3) and Lewis lung carcinoma cells expressing v5 epitope-tagged Ang-1 and Ang-2 were used in our studies. The results indicated that Ang-1 is secreted and incorporated into the ECM of the tumor cells, whereas Ang-2 is not associated with the ECM. The mutagenesis study indicated the domain that is responsible for the ECM association of Ang-1 is the linker peptide region between the coiled-coil and the fibrinogen-like domains. A weak binding between the coiled-coil domain of Ang-1 and the ECM was observed. Immunocytochemistry study revealed a distinct ECM distribution pattern of Ang-1, which is quite different from that of fibronectin, laminin, and collagen types I and IV. The ECM-associated Ang-1 proteins are released, and Tie-2 receptors are phosphorylated upon the adhesion of human umbilical vein endothelial cells. Implications of the difference in the ECM association of Ang-1 and Ang-2, which are related to the regulation of angiopoietin activity and their roles in local versus distant angiogenesis during tumor metastasis, are discussed.

Development of poly-( d, l-lactide–coglycolide) microsphere formulations containing recombinant human vascular endothelial growth factor to promote local angiogenesis

Although preclinical animal studies have demonstrated the utility of recombinant human vascular endothelial growth factor (rhVEGF) in promoting neovascularization in regions of ischemia, rhVEGF systemic administration did not provide clinical benefit to patients in recent placebo-controlled Phase II clinical trials. The amount of rhVEGF localized in the ischemic region after systemic administration is minimal and does not persist for more than 1 day. A greater persistence of rhVEGF at the region of ischemia may provide an increased angiogenesis with the eventual formation of patent blood vessels to restore nourishment to the tissues. We sought to develop a formulation of rhVEGF in poly( d, l-lactide–co-glycolide) (PLG) microspheres that would provide a continuous local delivery of intact protein. A stable formulation of rhVEGF for encapsulation contained a small amount of a stabilizing sugar, trehalose. Addition of excess trehalose increased the rate of release from the PLG. In addition, PLG with free acid end groups appeared to retard the initial release of rhVEGF by associating with it through ionic interactions at the positively charged heparin binding domain. rhVEGF was released continuously for 21 days with a very low (less than 10%) initial burst. The released rhVEGF aggregated and hydrolyzed over time and lost heparin affinity but not receptor affinity. The compression molding of rhVEGF PLG microspheres into disks yielded formulations with a low initial release and a lag of 10 days followed by complete release. The PLG microsphere formulations were assessed in the corneal implant model of angiogenesis and generated a dose-dependent angiogenic response. These formulations were also administered intravitreally and subretinally, generating local neovascularization comparable to the human disease states, vitroretinopathy and age-related macular degeneration, respectively. The rhVEGF PLG formulations may increase local angiogenesis without systemic side effects and may also be useful in the development of ocular disease models.

Inflammatory arthritis, hypoxia and vascularity

We have employed novel technology to investigate the relationship between synovial tissue oxygen levels and vascularity in human inflammatory arthritis. Silver microelectrodes were used to measure synovial tissue oxygen levels in knee joints of 15 patients with inflammatory arthritis (6 RA, 2 SLE, 1 psoriatic, 1 crystal, 2 reactive and 2 seronegative oligo-arthritides). Synovial membrane cells were obtained from tissue biopsies and ex-vivo production of vascular endothelial growth factor (VEGF) was measured. In RA patients, 50 ml N/Saline was injected into the joint and the electrode positioned in the cavity such that the rate of oxygen consumption could be measured. Microelectrodes were also used to assess synovial pO2 levels in a single metacarpophalngeal (MCP) joint in 5 RA patients. These joints were simultaneously imaged by high resolution ultrasound and power colour doppler to determine the relationship between joint architecture, vasculature and tissue pO2. In knees, synovial tissue pO2 levels were significantly lower in patients with active RA (mean = 7 mm Hg) than in patients without RA (mean = 40 mm Hg; P = 0.002). In RA, oxygen was consumed from N/Saline introduced into the cavity at a rate of 20.5 mm Hg/min. Production of VEGF from synovial cells was greater for patients with RA (mean = 868 pg/106 cells) than from synovial cells from patients without RA (mean = 84 pg/106 cells; P < 0.01). In the 5 MCP joints studied, a total of 9 vascular areas were sampled. The mean pO2 at these sites was 97 mmHg. In 19 non-vascular areas sampled, the mean pO2 was 34 mm Hg (range 6-73). In a vascular erosion the tissue pO2 was measured as 41 mmHg. In conclusion, marked hypoxia is observed in selected regions of inflamed synovium and is a likely stimulus for local VEGF production and angiogenesis. However, the increased vascularity associated with erosive damage is insufficient to restore oxygen homeostasis at the site of joint destruction.