Akt/protein kinase B and endothelial nitric oxide synthase mediate muscular neovascularization induced by tissue kallikrein gene transfer.
BACKGROUND
Angiogenesis gene therapy with human tissue kallikrein (hTK) has shown promise for ischemic disease. The present study was undertaken to (1) assess an optimal gene transfer modality, (2) clarify hTK angiogenic pathways, and (3) discount possible side effects.


RESULTS
The hTK gene was transferred to murine adductors by increasing doses of an adenovirus (Ad.hTK). Heterologous protein production was evaluated by ELISA and immunohistochemistry. Structural and functional characteristics of hTK-induced neovascularization were assessed. Muscular endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF)-A mRNA and protein content were evaluated by real-time polymerase chain reaction and Western blotting. The ability of hTK to phosphorylate-activate Akt/protein kinase B (Akt-B) and VEGF receptor 2 (VEGF-R2) was also determined. Implication of the aforementioned mechanisms in Ad.hTK-induced neovascularization was challenged by blocking Akt-B with a dominant-negative Akt construct; NOS with N(G)-nitro-L-arginine methyl ester; and VEGF-A with neutralizing antibody, VEGF-R2 antagonist, or Ad carrying soluble VEGF-R1 gene. We found that 10(7) PFU Ad.hTK led to peak increases in capillary and arteriole density. Newly developed arterioles persisted for up to 8 weeks. Ad.hTK did not change microvascular permeability. Ad.hTK upregulated eNOS mRNA and protein and activated Akt-B through Ser-473 phosphorylation. Inhibitory studies documented that these biochemical events were instrumental to Ad.hTK-induced neovascularization. In contrast, Ad.hTK neither affected VEGF-A and VEGF-R2 levels nor increased VEGF-R2 phosphorylation. Consistently, Ad.hTK-induced neovascularization was not disturbed by any of the different approaches used to block VEGF-A.


CONCLUSIONS
Our findings provide new information on the pathway involved in hTK-induced neoangiogenesis and represent an advancement toward clinical applications with Ad.hTK.
Emanueli
Costanza
C
Experimental Medicine and Gene Therapy Section, INBB, Alghero, Italy. emanueli@yahoo.com
Salis
Maria B
MB
Van Linthout
Sophie
S
Meloni
Marco
M
Desortes
Elisa
E
Silvestre
Jean-Sebastien
JS
Clergue
Michel
M
Figueroa
Carlos D
CD
Gadau
Sergio
S
Condorelli
Gianluigi
G
Madeddu
Paolo
P
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002455:Cell Division; D042783:Endothelial Cells / Q000166:cytology; D004730:Endothelium, Vascular / Q000166:cytology; D004790:Enzyme Induction / Q000187:drug effects; D005122:Exudates and Transudates; D005799:Genes, Dominant; D005822:Genetic Vectors / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008833:Microcirculation; D018482:Muscle, Skeletal / Q000098:blood supply* / Q000378:metabolism; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D018919:Neovascularization, Physiologic / Q000187:drug effects* / Q000235:genetics; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020840:Tissue Kallikreins / Q000235:genetics / Q000502:physiology*; D014161:Transduction, Genetic; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000032:analysis
D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D010768:Phosphoserine; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; D040301:Vascular Endothelial Growth Factor Receptor-2; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D020840:Tissue Kallikreins; D019331:NG-Nitroarginine Methyl Ester