Regression of Human Breast Carcinoma in Nude Mice after Adsflt Gene Therapy Is Mediated by Tumor Vascular Endothelial Cell Apoptosis.

Two vascular endothelial growth factor (VEGF) receptors, FLT-1 and KDR, are expressed preferentially in proliferating endothelium. There is increasing evidence that recombinant, soluble VEGF receptor domains interfering with VEGF signaling may inhibit in vivo neoangiogenesis, tumor growth and metastatic spread. We hypothesized that a soluble form of FLT-1 receptor (sFLT-1) could inhibit the growth of pre-established tumors via an anti-angiogenic mechanism. A replication-deficient adenovirus (Ad) vector carrying the sflt-1 cDNA (Adsflt) was used to overexpress the sFLT-1 receptor in a breast cancer animal model. MCF-7 cells, which produce VEGF, were used to establish solid tumors in the mammary fat pads of female nude mice. After six weeks, tumors were injected either with Adsflt or a negative control virus (AdCMV.ßgal). After six months, average tumor volume in the Adsflt-infected group (33 ± 22 mm3) decreased by 91% relative to that of the negative control group (388 ± 94 mm3; p < 0.05). Moreover, 10 of 15 Adsflt-infected tumors exhibited complete regression. The vascular density of Adsflt-infected tumors was reduced by 50% relative to that of negative controls (p < 0.05), which is consistent with sFLT-1-mediated tumor regression through an anti-angiogenic mechanism. Moreover, cell necrosis and fibrosis associated with long-term regression of Adsflt−infected tumors were preceded by apoptosis of tumor vascular endothelial cells. Mice treated with Adsflt intratumorally showed no delay in the healing of cutaneous wounds, providing preliminary evidence that Ad-mediated sFLT-1 overexpression may be an effective anti-angiogenic therapy for cancer without the risk of systemic anti-angiogenic effects.

Inhibition of angiogenesis in lipodermatosclerosis: implication for venous ulcer formation.

Lipodermatosclerosis refers to skin induration of the lower extremities characterized by tortuous, hyperpermeable vessels preceding venous leg ulcerations. Protein ligands and receptor tyrosine kinases that specifically regulate endothelial cell function are mainly involved in physiological as well as in disease-related angiogenesis. These ligand/receptor systems include the vascular endothelial growth factor (VEGF) and the angiopoietin (Ang) families and their receptor the tyrosine kinase with immunoglobulin-like domains (Tie-2) as well as the VEGF receptor family (VEGF-R1 and VEGF-R2). In the present study, the contribution of these endothelium-specific ligand/receptor systems in tissue samples of lipodermatosclerosis was evaluated. Our results provide evidence, that the mRNA-transcripts of VEGF (p<0.01), Ang-1 (p<0.1), Ang-2 (p<0.1) and VEGF-R1 (p<0.01) were significantly upregulated in all samples of lipodermatosclerosis in comparison with healthy skin by using reverse transcriptase-polymerase chain reaction. On protein level VEGF (p<0.01), Ang-1 (p<0.1), Ang-2 (p<0.1) and VEGF-R1 (p<0.01) were significantly elevated as well. Solely for Tie-2 and for VEGF-R2 no statistical difference could be detected on mRNA and protein level in patients with lipodermatosclerosis in comparison with healthy skin. By immunohistochemistry we confirmed upregulated protein expression for VEGF, Ang-1, Ang-2 and VEGF-R1 compared with healthy skin. Our findings strongly suggest that an imbalance between these ligand/receptor systems might contribute to the pathophysiology of advanced stages of chronic venous insufficiency. Inhibition of angiogenesis could significantly impact the tissue breakdown in lipodermatosclerosis and could hereby enable the formation of venous leg ulcerations.

Changes in serum soluble VEGFR-1 and Tie-2 receptors in colorectal cancer patients following surgical resections.

AIM: Determination of changes in serum levels of soluble (s) VEGFR-1 and Tie-2 receptors in colorectal cancer patients following resection in the search for novel tumour markers. PATIENTS AND METHODS: Forty-five patients with primary colorectal cancer and 29 normal subjects were recruited. Serum sVEGFR-1 and sTie-2 receptors were assayed using ELISA. RESULTS: sVEGFR-1 was detectable in 27% (10/37) and 12.5% (1/8) of cancer patients prior to curative and palliative resections, respectively, whilst 65.5% (19/29) of normal controls had detectable sVEGFR-1 levels. sTie-2 receptor levels were significantly raised in patients when compared with normal controls (p=0.0018). Furthermore, sTie-2 receptor levels were significantly higher in patients with metastases than those without (p=0.02). sTie-2 receptors demonstrated a significant drop in patients undergoing both curative (p<0.0001) and palliative resections (p=0.012). CONCLUSION: sVEGFR-1 levels were suppressed and sTie-2 receptor levels were raised in colorectal cancer patients. This data supports the potential use of sTie-2 receptor as a tumour marker.

Angiogenic factors in squamous cell carcinoma of the oral cavity: do they have prognostic relevance?

INTRODUCTION: Angiogenesis is a cascade-like mechanism which is essential for tumour growth and metastasis. Therefore the existence of angiogenic molecules and the density of activated endothelial cells in individual tumours is of major interest. MATERIAL/PATIENTS: In order to evaluate the prognostic significance of these molecules, the distribution pattern was studied of vascular endothelial growth factor (VEGF) and activated endothelial cells in tumours and normal, healthy oral mucosal specimens from 51 consecutive patients with primary oral squamous cell carcinoma. STUDY DESIGN: Frozen sections (vascular endothelial growth factor) and paraffin-embedded sections (endoglin, CD105) were investigated quantitatively by immunohistochemistry. The Pearson correlation, the non-parametric Mann-Whitney test, the non-parametric Wilcoxon rank sum test with multiple comparisons and the non-parametric Kruskal-Wallis test with multiple comparisons were used for statistical analyses. RESULTS: Endoglin expression in tumour tissue was significantly higher than in normal healthy mucosa (P<0.001). T1 tumours showed a significantly lower staining for endoglin compared with T2, T3 and T4 tumours but there was no increase with each T stage. No statistical correlation was found between VEGF expression and endoglin staining. CONCLUSIONS: Even though there is controversy about the prognostic relevance of VEGF, our results suggest that the factor is not suitable to decide prognosis in oral cancer. Endoglin may have a significant role in the development of squamous cell carcinoma of the oral cavity and might be relatively more specific than commonly used endothelial markers.

Implications of vascular endothelial growth factor, sFlt-1, and sTie-2 in plasma, serum and cerebrospinal fluid during cerebral ischemia in man.

The relation between cerebral ischemia and local release of angiogenic factors was investigated after subarachnoid hemorrhage (SAH) in humans. Time-dependent concentration-changes of vascular endothelial growth factor (VEGF), sFlt-1 and sTie-2 extracted from plasma, serum, and cerebrospinal fluid (ventricular, cisternal, and lumbar) were analyzed in 15 patients surgically treated for ruptured aneurysms of the anterior circulation (Hunt and Hess grades I-V). Data were related to brain Po2 (Pbro2) and cerebral energy metabolites (extracellular lactate, pyruvate, glutamate, and glycerin concentrations) as well as clinical and radiologic reference data. Delayed impairment of cerebral perfusion secondary to progressive microcirculatory alterations was associated with reduced local Pbro2 and energy metabolism (increased lactate-pyruvate ratio, glutamate and glycerine levels). Elevated serum/plasma and CSF concentrations of VEGF, sFlt-1, and sTie-2 matched the scale of ischemic tissue hypoxia. Excessive VEGF/sFlt-1 and sTie-2 levels were related to Pbro2 values consistently less than 5 mm Hg, glutamate concentrations greater than 300 micromol/L, lactate-pyruvate ratio greater than 300, cerebral infarction, and reduced outcome (P < 0.01). Delayed microcirculatory impairment was mirrored by distinct elevation of cisternal and arterial VEGF and sFlt-1 concentrations, suggesting local induction of angiogenesis. Arterial levels of VEGF, sFlt-1, and sTie-2 reflect both extent and time course of compensatory, yet clinically inefficient, angiogenesis in the absence of general hypoxia.