Mariarosaria Galeano

Adeno-associated viral vector-mediated human vascular endothelial growth factor gene transfer stimulates angiogenesis and wound healing in the genetically diabetic mouse.

Aims/hypothesisWe studied the gene therapy efficacy of diabetes-associated wound healing disorder with an adeno-associated virus (AAV) vector expressing the 165-amino acid isoform of human vascular endothelial growth factor-A (VEGF-A) by using an incisional skin-wound model produced on the back of female diabetic C57BL/KsJ db+/db+ mice and their normal littermates (db+/+m).MethodsAnimals were randomized to receive intradermally into the wound edges either rAAV-LacZ (a control gene), or rAAV-VEGF165. Animals were killed on different days (7 and 14 days after skin injury) and wounded skin tissues were used for gene marker studies, histological evaluation and immunohistochemistry, and wound breaking strength analysis. Furthermore we studied the VEGF mature protein in the wounds.ResultsWe found that AAV vectors are highly efficient for gene transfer to the mouse skin, displaying an exquisite tropism for the panniculus carnosus by using the beta-galactosidase activity assay. We confirmed the increased expression of the angiogenic factor at day 7 by measuring the wound content of the mature protein. Delivery of VEGF165 to incisional skin wounds of diabetic mice resulted in a remarkable induction of new vessel formation with consequent improvement in the wound healing process. The rAAV-VEGF165 gene improved wound healing in diabetic mice through the stimulation of angiogenesis, reepithelization, synthesis and maturation of extracellular matrix. Moreover the recombinant AAV encoding the human VEGF165 increased the breaking strenght of the wound and enhanced the wound content of VEGF.Conclusion/interpretationOur study suggests that VEGF gene transfer might represent a new approach to treat wound healing disorders associated with diabetes.

Recombinant human erythropoietin improves angiogenesis and wound healing in experimental burn wounds

Objective:Erythropoietin interacts with vascular endothelial growth factor (VEGF) and stimulates endothelial cell mitosis and motility; thus it may be of importance in the complex phenomenon of wound healing. The purpose of this study was to investigate the effect of recombinant human erythropoietin (rHuEPO) on experimental burn wounds. Design:Randomized experiment. Setting:Research laboratory. Subjects:C57BL/6 male mice weighing 25–30 g. Interventions:Mice were immersed in 80°C water for 10 secs to achieve a deep-dermal second degree burn. Animals were randomized to receive either rHuEPO (400 units/kg/day for 14 days in 100 &mgr;L subcutaneously) or its vehicle alone (100 &mgr;l/day distilled water for 14 days subcutaneously). On day 14 the animals were killed. Burn areas were used for histologic examination, evaluation of neoangiogenesis by immunohistochemistry, and expression (Western blot) of the specific endothelial marker CD31 as well as quantification of microvessel density, measurement of VEGF wound content (enzyme-linked immunosorbent assay), expression (Western blot) of endothelial and inducible nitric oxide synthases, and determination of wound nitric oxide (NO) products. Measurements and Main Results:rHuEPO increased burn wound reepithelialization and reduced the time to final wound closure. These effects were completely abated by a passive immunization with specific antibodies against erythropoietin. rHuEPO improved healing of burn wound through increased epithelial proliferation, maturation of the extracellular matrix, and angiogenesis. The hematopoietic factor augmented neoangiogenesis as suggested by the marked increase in microvessel density and by the robust expression of the specific endothelial marker CD31. Furthermore, rHuEPO enhanced the wound content of VEGF caused a marked expression of endothelial and inducible nitric oxide synthases and increased wound content of nitric oxide products. Conclusions:Our study suggests that rHuEPO may be an effective therapeutic approach to improve clinical outcomes after thermal injury.

Recombinant human erythropoietin stimulates angiogenesis and healing of ischemic skin wounds.

Wound healing in ischemic tissues such as flap margins due to inadequate blood supply is still a source of considerable morbidity in surgical practice. Adequate tissue perfusion is particularly important in wound healing. We investigated the effects of recombinant human erythropoietin (rHuEPO) on wound healing in an ischemic skin wound model. Sixty-three Sprague-Dawley rats were used. Normal incisional wound and H-shaped double flaps were used as the wound models. Animals were treated with rHuEPO (400 IU/kg) or its vehicle. Rats were killed on different days (3, 5, and 10 days after skin injury) and the wounded skin tissues were used for immunohistochemistry and for analysis of vascular endothelial growth factor content and collagen content. Tissue transglutaminase immunostaining of histological specimens was used as a vascular marker to determine the level of microvessel density. The results showed a higher level of vascular endothelial growth factor protein and an increased microvessel density in ischemic wounds with rHuEPO treatment than the normal incisional wounds and ischemic control wounds. Collagen content was higher in the incisional wounds and in the ischemic wounds with rHuEPO treatment compared with the ischemic control wounds. Our results suggest that erythropoietin may be an effective therapeutic approach in improving healing in ischemic skin wounds.

Attenuated Cerulein-Induced Pancreatitis in Nuclear Factor–κB–Deficient Mice

Nuclear factor (NF)-κB plays a central role in acute pancreatitis. We studied cerulein (CER)-induced pancreatitis in NF-κB knockout (KO) mice. NF-κB KO mice and normal control littermate wild-type (WT) mice were given four hyperstimulating doses of cerulein every hour to elicit secreatagogue-induced pancreatitis. Malonildialdehyde activity, glutathione levels, myeloperoxidase activity, TNF-α, and NF-κB binding activity and its inhibitory protein IκBα were studied in the pancreas. Furthermore, we measured plasma lipase and amylase and the histological damage. KO mice had reduced malonildialdehyde levels (WT + CER = 4.083 ± 0.95 μmol/g; KO + CER = 1.513 ± 0.63 μmol/g), decreased myeloperoxidase activity (WT + CER = 19.3 ± 2.39 mU/g; KO + CER = 10.21 ± 2.05 mU/g), increased glutathione levels (WT + CER 6.22 ± 2.46 μmol/g; KO + CER = 15. 516 ± 2.92 μmol/g), and reduced serum levels of amylase (WT + CER = 2519 ± 656.9 U/L; KO + CER = 916 ± 280.4 U/L) and lipase (WT + CER = 1420 ± 170 U/L; KO + CER = 861 ± 172. 3 U/L). KO mice showed reduced pancreatic NF-κB activation, decreased TNF-α tissue content, and reduced histologic alterations. Our data suggest that KO mice have an attenuated cerulein-induced pancreatitis and help to define the possible interaction between NF-κB activation and oxidative stress in this deleterious event.

Simvastatin enhances VEGF production and ameliorates impaired wound healing in experimental diabetes

Statins have different effects beyond cholesterol reduction and stimulate angiogenesis. We investigated the effect of simvastatin in diabetes-related healing defects. An incisional skin wound model produced on the back of female diabetic mice (db(+)/db(+)) and their normoglycemic littermates (db(+)/(+)m) was used. Animals were treated daily either with simvastatin (5 mg/(kgi.p.)) or vehicle. Mice were killed on different days (3, 6 and 12 after skin injury) for measurement of vascular endothelial growth factor (VEGF) mRNA and protein expression, to assess histologically the healing process and to evaluate wound breaking strength and angiogenesis by CD31 immunostaining. Simvastatin administration in diabetic mice increased VEGF mRNA (simvastatin=4.8+/-0.6n-fold/beta-actin; vehicle=2.3+/-0.4n-fold/beta-actin) and protein expression (simvastatin=5+/-0.7 integrated intensity; vehicle=2.2+/-0.3 integrated intensity) and enhanced nitric oxide wound content at day 6. Additionally, the statin augmented breaking strength and PECAM-1 immunostaining at day 12. Finally, simvastatin administration restored the impaired wound healing process in diabetic mice. Similar results were obtained in normoglycaemic mice. Passive immunization with anti-VEGF antibody (10 microg/mouse) completely abrogated the beneficial effects of simvastatin on healing in diabetic mice. Simvastatin has potential application in diabetes-related wound healing disorders.

Oxidative stress causes nuclear factor-κB activation in acute hypovolemic hemorrhagic shock

Nuclear Factor kappaB (NFkappaB) is an ubiquitous rapid response transcription factor involved in inflammatory reactions and exerts its action by expressing cytokines, chemokines, and cell adhesion molecules. We investigated the role of NF-kappaB in acute hypovolemic hemorrhagic (Hem) shock. Hem shock was induced in male anesthetized rats by intermittently withdrawing blood from an iliac catheter over a period of 20 min (bleeding period) until mean arterial blood pressure (MAP) fell and stabilized within the range of 20-30 mmHg. Hemorrhagic shocked rats died in 26.3 +/- 2.1 min following the discontinuance of bleeding, experienced a marked hypotension (mean arterial blood pressure = 20-30 mmHg) and had enhanced plasma levels of Tumor Necrosis Factor-alpha (200 +/- 15 pg/ml, 20 min after the end of bleeding). Furthermore, aortas taken 20 min after bleeding from hemorrhagic shocked rats showed a marked hypo-reactivity to phenylephrine (PE; 1nM to 10 microM) compared with aortas harvested from sham shocked rats. Hem shocked rats also had increased levels of TNF-alpha mRNA in the liver (15-20 min after the end of bleeding) and enhanced plasma levels of 2,5-dihydroxybenzoic acid (2,5-DHBA; 6 +/- 2.2 microm), 2,3-dihydroxybenzoic acid (2,3-DHBA; 13 +/- 2.1 microm), both studied to evaluate OH(*) production. Electrophoretic mobility shift assay showed that liver NF-kappaB binding activity increased in the nucleus 10 min after the end of hemorrhage and remained elevated until the death of animals. Western blot analysis suggested that the levels of inhibitory IkappaBalpha protein in the cytoplasm became decreased at 5 min after the end of bleeding. IRFI-042, a vitamin E analogue (20 mg/kg intraperitoneally 2 min after the end of bleeding), inhibited the loss of IkappaBalpha protein from the cytoplasm and blunted the increase in NF-kappaB binding activity. Furthermore IRFI-042 increased survival time (117.8 +/- 6.51 min; p <.01) and survival rate (vehicle = 0% and IRFI-042 = 80%, at 120 min after the end of bleeding), reverted the marked hypotension, decreased liver mRNA for TNF-alpha, reduced plasma TNF-alpha (21 +/- 4.3 pg/ml), and restored to control values the hypo-reactivity to PE. Our results suggest that acute blood loss (50% of the estimated total blood volume over a period of 20 min) causes early activation of NF-kappaB, likely through an increased production of reactive oxygen species. This experiment indicates that NF-kappaB-triggered inflammatory cascade becomes early activated during acute hemorrhage even in the absence of resuscitation procedures.

Polydeoxyribonucleotide stimulates angiogenesis and wound healing in the genetically diabetic mouse

Healing of diabetic wounds still remains a critical medical problem. Polydeoxyribonucleotide (PDRN), a compound having a mixture of deoxyribonucleotide polymers, stimulates the A2 purinergic receptor with no toxic or adverse effect. We studied the effects of PDRN in diabetes‐related healing defect using an incisional skin‐wound model produced on the back of female diabetic mice (db+/db+) and their normal littermates (db+/+m). Animals were treated daily for 12 days with PDRN (8 mg/kg/ip) or its vehicle (100 μL 0.9%NaCl). Mice were killed 3, 6, and 12 days after skin injury to measure vascular endothelial growth factor (VEGF) mRNA expression and protein synthesis, to assay angiogenesis and tissue remodeling through histological evaluation, and to study CD31, Angiopoietin‐1 and Transglutaminase‐II. Furthermore, we measured wound breaking strength at day 12. PDRN injection in diabetic mice resulted in an increased VEGF message (vehicle=1.0±0.2 n‐fold vs. β‐actin; PDRN=1.5±0.09 n‐fold vs. β‐actin) and protein wound content on day 6 (vehicle=0.3±0.07 pg/wound; PDRN=0.9±0.1 pg/wound). PDRN injection improved the impaired wound healing and increased the wound‐breaking strength in diabetic mice. PDRN also caused a marked increase in CD31 immunostaining and induced Transglutaminase‐II and Angiopoietin‐1 expression. Furthermore, the concomitant administration of 3,7‐dimethyl‐1‐propargilxanthine, a selective adenosine A2A receptor antagonist, abolished PDRN positive effects on healing. However, 3,7‐dimethyl‐1‐propargilxanthine alone did not affect wound healing in both diabetic mice and normal littermates. These results suggest that PDRN might be useful in wound disorders associated with diabetes.

Effect of recombinant adeno-associated virus vector-mediated vascular endothelial growth factor gene transfer on wound healing after burn injury.

ObjectiveThe purpose of this study was to investigate the effect of recombinant adeno-associated viral (rAAV) vector-mediated human vascular endothelial growth factor (VEGF165) transfer on experimental burn wounds. DesignRandomized experiment. SettingResearch laboratory. SubjectsC57BL/6 male mice weighing 25–30 g. InterventionsMice were immersed in 80°C water for 10 secs to achieve a partial-thickness scald burn. Animals were randomized to receive at two injection sites on the edge of the burn either 1011 copies of the rAAV-VEGF165 or the vector carrying the control and inert gene &bgr;-galactosidase (rAAV-LacZ). On day 14 the animals were killed. Burn areas were used for histologic examination, evaluation of VEGF expression (immunohistochemistry) and VEGF wound content (enzyme-linked immunosorbent assay), determination of wound nitrite, and measurement of messenger RNA (mRNA) for endothelial and inducible nitric oxide synthase (eNOS and iNOS). Measurements and Main ResultsrAAV-VEGF165 increased epithelial proliferation, angiogenesis, and maturation of the extracellular matrix. Furthermore, gene transfer enhanced VEGF expression, studied by immunohistochemistry, and the wound content of the mature protein (rAAV-LacZ, 11 ± 5 pg/wound; rAAV-VEGF165, 104 ± 7 pg/wound). Moreover, VEGF165 gene transfer increased wound content of nitrate. Finally, rAAV-VEGF165 administration enhanced the messenger RNA for eNOS (rAAV-VEGF165, 1.1 ± 0.2 relative amount of eNOS mRNA; rAAV-LacZ, 0.66 ± 0.3 relative amount of eNOS mRNA) and iNOS (rAAV-VEGF165, 0.8 ± 0.09 relative amount of iNOS mRNA; rAAV-LacZ, 0.45 ± 0.05 relative amount of iNOS mRNA). ConclusionOur study suggests that rAAV-VEGF gene transfer may be an effective therapeutic approach to improve clinical outcomes after thermal injury.

Systemic administration of high-molecular weight hyaluronan stimulates wound healing in genetically diabetic mice.

Hyaluronic acid (HA), an essential component of the extracellular matrix, is an efficient space filler that maintains hydration, serves as a substrate for assembly of proteoglycans and is involved in wound healing. Although numerous pieces of evidence demonstrate beneficial effects in promoting wound healing in diabetes, a systemic approach has never been tested. We used an incisional wound healing model in genetically diabetic mice to test the effects of systemic injection of HA. Diabetic (n=56) and normoglycemic (n=56) mice were subjected to incision and randomized (8 groups of 7 animals each) to receive HA at different doses, 7.5, 15 and 30mg/kg/i.p., or vehicle (0.9% NaCl solution) for 12days. At the end of the experiment animals were sacrificed and skin wounds were excised for histological, biochemical and molecular analysis. Histology revealed that the most effective dose to improve wound repair and angiogenesis in diabetic mice was 30mg/kg. Furthermore HA injection (30mg/kg) improved the altered healing pattern in diabetic animals, increased skin remodeling proteins TGF-β and transglutaminase-II and restored the altered expression of cyclin B1/Cdc2 complex. Evaluation of skin from diabetic animals injected with HA revealed also an increase in HA content, suggesting that systemic injection may be able to restore the reduced intracellular HA pool of diabetic mice. Finally HA markedly improved skin mechanical properties. These promising results, if confirmed in a clinical setting, may improve the care and management of diabetic patients.

Polydeoxyribonucleotide improves angiogenesis and wound healing in experimental thermal injury

Objective:Polydeoxyribonucleotide contains a mixture of nucleotides and interacts with adenosine receptors, stimulating vascular endothelial growth factor expression and wound healing. The purpose of this study was to investigate the effect of polydeoxyribonucleotide on experimental burn wounds. Design:Randomized experiment. Setting:Research laboratory at a university hospital. Subjects:Thermal injury in mice. Interventions:Mice were immersed in 80°C water for 10 secs to achieve a deep-dermal second-degree burn. Animals were randomized to receive either polydeoxyribonucleotide (8 mg/kg/day intraperitoneally for 14 days) or its vehicle alone (0.9% NaCl solution at 100 μL/day intraperitoneally). On days 7 and 14 the animals were killed. Blood was collected for tumor necrosis factor-α measurement; burn areas were used for histologic and immunohistochemical examination, for the evaluation of vascular endothelial growth factor and nitric oxide synthases by Western blot, and for the determination of wound nitric oxide products. Measurements and Main Results:Polydeoxyribonucleotide increased burn wound re-epithelialization and reduced the time to final wound closure. Polydeoxyribonucleotide improved healing of burn wound through increased epithelial proliferation and maturation of the extracellular matrix as confirmed by fibronectin and laminin immunostaining. Polydeoxyribonucleotide also improved neoangiogenesis as suggested by the marked increase in microvessel density and by the robust expression of platelet-endothelial cell adhesion molecule-1. Furthermore, polydeoxyribonucleotide blunted serum tumor necrosis factor-α and enhanced inducible nitric oxide synthase and vascular endothelial growth factor expression and the wound content of nitric oxide products. Conclusions:Our study suggests that polydeoxyribonucleotide may be an effective therapeutic approach to improve clinical outcomes after thermal injury.