In ovo administration of human recombinant leptin shows dose dependent angiogenic effect on chicken chorioallantoic membrane

BACKGROUND: Leptin, the cytokine produced by white adipose tissue is known to regulate food energy homeostasis through its hypothalamic receptor. In vitro studies have demonstrated that leptin plays a major role in angiogenesis through binding to the receptor Ob-R present on ECs by stimulating and initiating new capillary like structures from ECs. Various in vivo studies indicate that leptin has diverse effect on angiogenesis. A few reports have showed that leptin exerts pro angiogenic effects while some suggested that it has antiangiogenic potential. It is theoretically highly important to understand the effect of leptin on angiogenesis to use as a therapeutic molecule in various angiogenesis related pathological conditions. Chicken chorio allantoic membrane (CAM) on 9th day of incubation was incubated with 1, 3 and 5 µg concentration of HRL for 72 h using gelatin sponge. Images where taken after every 24 h of incubation and analysed with Angioguant software. The treated area was observed under microscope and histological evaluation was performed for the same. Tissue thickness was calculated morphometrically from haematoxylin and eosin stained cross sections. Reverse transcriptase PCR and immunohistochemistry were also performed to study the gene and protein level expression of angiogenic molecules. RESULTS: HRL has the ability to induce new vessel formation at the treated area and growth of the newly formed vessels and cellular morphological changes occur in a dose dependent manner. Increase in the tissue thickness at the treated area is suggestive of initiation of new capillary like structures. Elevated mRNA and protein level expression of VEGF165 and MMP2 along with the activation of ECs as demonstrated by the presence of CD34 expression supports the neovascularization potential of HRL. CONCLUSION: Angiogenic potential of HRL depends on the concentration and time of incubation and is involved in the activation of ECs along with the major interaction of VEGF 165 and MMP2. It is also observed that 3 µg of HRL exhibits maximum angiogenic potential at 72 h of incubation. Thus our data suggest that dose dependent angiogenic potential HRL could provide a novel role in angiogenic dependent therapeutics such as ischemia and wound healing conditions.

Terapia gênica com VEGF para angiogênese na angina refratária: ensaio clínico fase I/II / VEGF gene therapy for angiogenesis in refractory angina: phase I/II clinical trial

OBJETIVO: Avaliar a segurança, viabilidade e efeitos iniciais, clínicos e sobre a perfusão miocárdica, da administração intramiocárdica, transtorácica, de VEGF 165 plasmidial em pacientes com doença arterial coronariana avançada e angina refratária, não passíveis de revascularização percutânea e cirúrgica. MÉTODOS: Ensaio clínico fase I/II. Treze pacientes cardiopatas isquêmicos com angina refratária apesar de tratamento medicamentoso máximo por no mínimo seis meses, não passíveis de revascularização cirúrgica ou por cateter foram submetidos a injeções intramiocárdicas de 2000µg VEGF 165 plasmidial. Os pacientes foram avaliados por cintilografia miocárdica, teste ergométrico, questionário de qualidade de vida (Minnesota) e determinação das classes de insuficiência cardíaca (NYHA) e angina (CCS). RESULTADOS: Não houve óbitos ou reintervenções. Durante o período de tratamento medicamentoso máximo, não se observou diferenças em cintilografias miocárdicas, testes ergométricos e questionários de qualidade de vida, ainda, houve tendência a piora das classes NYHA (P=0,05) e CCS (P=0,05). Três meses após intervenção, observou-se melhora dos escores cintilográficos SSS (18,38±7,51 vs. 15,31±7,29, P=0,003) e SRS (11,92±7,49 vs. 8,53±6,68, P=0,002), porém não na proporção da extensão da área de miocárdio isquêmico (23,38±13,12 por cento vs. 20,08±13,88 por cento, P=0,1). Houve tendência a melhora dos METs nas ergometrias (7,66±4,47 vs. 10,29±4,36, P=0,08), melhora do escore de qualidade de vida (48,23±18,35 vs. 30,15±20,13; P=0,02) e das classes NYHA (3,15±0,38 vs. 1,77±0,83, P=0,001) e CCS (3,08±0,64 vs. 1,77±0,83, P=0,001), no mesmo período. CONCLUSÕES: A terapia demonstrou-se segura e viável nesta série de pacientes. Os resultados iniciais tendem a demonstrar melhora na gravidade da angina e redução da intensidade da isquemia miocárdica.

OBJECTIVE: Safety, feasibility and early myocardial angiogenic effects evaluation of transthoracic intramyocardial phVEGF165 administration for refractory angina in no option patients. METHODS: Cohort study, in which 13 patients with refractory angina under optimized clinical treatment where included, after cineangiograms had been evaluated and found unfeasible by surgeon and interventional cardiologist. Intramyocardial injections of 5mL solution containing plasmidial VEGF165 where done over the ischemic area of myocardium identified by previous SPECT/Sestamibi scan. Evaluations included a SPECT scan, stress test, Minnesotta QOL questionnaire and NYHA functional class and CCS angina class determinations. RESULTS: There were no deaths or new interventions during the study period. There were no significant variations in SPECT scans, QOL scores and stress tests results during medical treatment in the included patients. After the 3rd post operative month, there was improvement in SPECT segmental scores, SSS (18.38±7.51 vs. 15.31±7.29, P=0.003) and SRS (11.92±7.49 vs. 8.53±6.68, P=0.002). The ischemic area extension, however, had non-significant variation (23.38±13.12 percent vs. 20.08±13.88 percent, P=0.1). Stress tests METs varied from 7.66±4.47 pre to 10.29±4.36 METs post-op (P=0.08). QOL score improved from 48.23±18.35 pre to 30.15±20.13 post-op points (P=0.02). NYHA class was 3.15±0.38 pre vs. 1.77±0.83 post-op (P=0.001) and angina CCS class, 3.08±0.64 vs. 1.77±0.83 (P=0.001). CONCLUSIONS: Intramyocardial VEGF165 therapy for refractory angina, in this small trial of no option patients, resulted feasible and safe. Early clinical and scintilographic data showed improvements in symptoms and myocardial perfusion, with regression of ischemia severity in treated areas.

Induction of inflammatory cell accumulation using human mast cell tryptases.

The aim of this study was to investigate the effects of human tryptases on inflammatory cell accumulation in vivo. Various concentrations of purified lung or skin tryptases were injected into the peritoneum of BALB/c mouse. At 6 hours or 16 hours following injection, cells from the peritoneal lavage were collected and stained with modified Wright's stain. Differential cell counts were performed and results were expressed as absolute numbers of each cell type per mouse peritoneum. The results showed a dose-dependent infiltration of neutrophils with a maximal increase of up to 32 fold or 43 fold in numbers at 16 hours following an injection of skin and lung tryptases, respectively. Skin tryptase was able to attract more eosinophils than lung tryptase. Significant increases in lymphocyte and macrophage numbers were also observed. In conclusion, both skin and lung tryptases are able to induce nucleated cell accumulation in the peritoneum of mice with similar specificity and potency.