Differential expression of VEGF121, VEGF165 and VEGF189 in angiomas and squamous cell carcinoma cell lines of the head and neck.

Vascular endothelial growth factor A (VEGF) is one of the major regulators of angiogenesis. It plays an important role during the process of physiological and pathological neovascularization. Variant VEGF isoforms are generated as a result of alternative pre-mRNA splicing. To determine the expression of VEGF isoforms in angioma and head and neck squamous cell carcinoma (HNSCC), both being dependent on pathological neovascularization, we included 11 HNSCC cell lines, 4 hemangiomas and 5 vascular malformations (VMs) in the study. Tonsil mucosa served as normal control. Using reverse transcription polymerase chain reaction (RT-PCR) sequencing, the VEGF isoforms VEGF(189), VEGF(165) and VEGF(121) were regularly detected in all tested samples. VEGF(121) was the most abundant isoform in all tested tissues, whereas VEGF(165) exhibited lower levels, and VEGF(189) only very small amounts of transcript. Interestingly, VMs expressed significantly higher (p=0.0286) amounts of VEGF(121) compared with hemangiomas, which had levels similar to normal control mucosa. HNSCC cell lines demonstrated on-average higher levels of all three isoforms compared with the controls. Consistent with the clinical staging, a trend for VEGF overexpression was observed in tumor cells derived from N+ tumors compared to those derived from N0 tumors. One drawback of this study was the small number of specimens available, particularly since VMs and hemangiomas are relatively rare diseases. Future studies need to follow-up on these observations and further evaluate the potential role of specific VEGF isoforms in the pathogenesis of hemangioma and VM.

A basal-cell-like compartment in head and neck squamous cell carcinomas represents the invasive front of the tumor and is expressing MMP-9.

Head and neck squamous cell carcinomas (HNSCCs) are the most frequent malignancies of the upper aerodigestive tract. The cancer stem cell (CSC) hypothesis concludes that CSCs constitute the dangerous tumor cell population due to their ability of self-renewal and being associated with relapse of tumor disease, invasiveness and resistance to chemo(radio)therapy. The aim of this study was to look for CSC candidates and expression of MMP-9 that previously was implicated in HNSCC invasiveness. Immunohistochemical, immunofluorescence and Western blot analysis were performed on HNSCC tumor specimens using antibodies specific for MMP-9, CD44, ALDH1 and CK14. Gelatinolytic activity was assessed by zymography. Pearson correlation analysis was used for statistical comparison. Immunohistochemical analysis found CD44 and MMP-9 to co-localize in tumor cells at the invasive front. Western blot analysis demonstrated a significant correlation (p=0.0047) between CD44 and MMP-9 in the tested tissues. In addition gelatinolytic activity of HNSCC tissues was found to significantly correlate (p=0.0010) with MMP-9 expression. The CD44(+) invasive front of the tumor was also positive for ALDH1 and CK14, all of them being typically expressed by cells in the basal cell layer of normal stratified squamous epithelia that also harbors the epithelial stem cells. The observations point to a role of a MMP-9 positive basal-cell-like cell layer in the process of HNSCC invasiveness. This compartment likely contains CSCs since it is expressing the putative CSC markers CD44, ALDH1 and CK14. This cell layer therefore should be considered a major therapeutic target in the treatment of head and neck cancer.

Recombinant adeno-associated virus-based gene transfer of cathelicidin induces therapeutic neovascularization preferentially via potent collateral growth.

Therapeutic neovascularization is a concept well validated in animal models, however, without clear-cut success in clinical studies. To achieve prolonged transgene expression, recombinant adeno-associated virus (rAAV) was used in a chronic ischemic hind-limb model and the human antimicrobial peptide cathelicidin (LL-37/hCAP-18) was used as proangiogenic factor. Seven days after femoral artery excision, 0.5 x 10(11) rAAV particles encoding for green fluorescent protein (rAAV.GFP), cathelicidin (rAAV.cath), or vascular endothelial growth factor A (rAAV.VEGF-A) were retroinfused into the anterior tibial vein of rabbits (n = 5 per group). In addition, one rAAV.cath-treated group obtained a constant infusion with the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin into the ischemic tissue starting on day 7. On day 7 and day 35 angiography of both hind limbs was performed for collateral quantification and frame count score (cinedensitometry). Capillary-to-muscle fiber ratios were obtained on day 35. Compared with controls, application of rAAV.cath induced a gain of perfusion (153 +/- 12 vs. 107 + 9% of day 7 controls) via increased collateral growth (length index, 161 +/- 14 vs. 97 +/- 9%, controls), but no significant capillary growth (1.16 +/- 0.09 vs. 0.99 +/- 0.08, controls). Wortmannin application completely abolished the effects of rAAV.cath, indicating the involvement of the PI3K signal pathway. In conclusion, rAAV-mediated cathelicidin expression is capable of inducing functionally relevant neovascularization, preferentially by collateral growth. The rAAV-based vectors as long-expressing vector expression systems and cathelicidin as proangiogenic factor provide a promising new combination in the treatment of peripheral artery disease.