Gold nanoparticle intratesticular injections as a potential animal sterilization tool: Long-term reproductive and toxicological implications.

This study aimed to evaluate the gold nanoparticles (AuNPs) animal sterilizing potential after intratesticular injections and long-term adverse reproductive and systemic effects. Adult male Wistar rats were divided into control and gold nanoparticle (AuNPs) groups. The rats received 200 µL of saline or AuNPs solution (16 µg/mL) on experimental days 1 and 7 (ED1 and ED7). After 150 days, the testicular blood flow was measured, and the rats were mated with females. After mating, male animals were euthanized for histological, cellular, and molecular evaluations. The female fertility indices and fetal development were also recorded. The results indicated increased blood flow in the testes of treated animals. Testes from treated rats had histological abnormalities, shorter seminiferous epithelia, and oxidative stress. Although the sperm concentration was lower in the AuNP-treated rats, there were no alterations in sperm morphology. Animals exposed to AuNPs had decreased male fertility indices, and their offspring had lighter and less efficient placentas. Additionally, the anogenital distance was longer in female fetuses. There were no changes in the histology of the kidney and liver, the lipid profile, and the serum levels of LH, testosterone, AST, ALT, ALP, albumin, and creatinine. The primary systemic effect was an increase in MDA levels in the liver and kidney, with only the liver experiencing an increase in CAT activity. In conclusion, AuNPs have a long-term impact on reproduction with very slight alterations in animal health. The development of reproductive biotechnologies that eliminate germ cells or treat local cancers can benefit from using AuNPs.

Acute toxicity and antitumor potential of 1,3,4-trisubstituted-1,2,3-triazole dhmtAc-loaded liposomes on a triple-negative breast cancer model.

For the first time, compounds developed from the 1,2,3-triazole scaffold were evaluated as novel drugs to treat triple-negative breast cancer (TNBC). Four organic salts were idealized as nonclassical bioisosteres of miltefosine, which is used in the topical treatment for skin metastasizing breast carcinoma. Among them, derivative dhmtAc displayed better solubility and higher cytotoxicity against the human breast adenocarcinoma cell line and mouse 4T1 cell lines, which are representatives of TNBC. In vitro assays revealed that dhmtAc interferes with cell integrity, confirmed by lactate dehydogenase leakage. Due to its human peripheral blood mononuclear cell (PBMC) toxicity, dhmtAc in vivo studies were carried out with the drug incorporated in a long-circulating and pH-sensitive liposome (SpHL-dhmtAc), and the acute toxicity in BALB/c mice was determined. Free dhmtAc displayed cardiac and pulmonary toxicity after the systemic administration of 5 mg/kg doses. On the other hand, SpHL-dhmtAc displayed no toxicity at 20 mg/kg. The in vivo antitumor effect of SpHL-dhmtAc was investigated using the 4T1 heterotopic murine model. Intravenous administration of SpHL-dhmtAc reduced the tumor volume and weight, without interfering with the body weight, compared with the control group and the dhmtAc free form. The incorporation of the triazole compound in the liposome allowed the demonstration of its anticancer potential. These findings evidenced 1,3,4-trisubstituted-1,2,3-triazole as a promising scaffold for the development of novel drugs with applicability for the treatment of patients with TNBC.

Catalyst-Free Click Chemistry for Engineering Chondroitin Sulfate-Multiarmed PEG Hydrogels for Skin Tissue Engineering.

The quest for an ideal biomaterial perfectly matching the microenvironment of the surrounding tissues and cells is an endless challenge within biomedical research, in addition to integrating this with a facile and sustainable technology for its preparation. Engineering hydrogels through click chemistry would promote the sustainable invention of tailor-made hydrogels. Herein, we disclose a versatile and facile catalyst-free click chemistry for the generation of an innovative hydrogel by combining chondroitin sulfate (CS) and polyethylene glycol (PEG). Various multi-armed PEG-Norbornene (A-PEG-N) with different molecular sizes were investigated to generate crosslinked copolymers with tunable rheological and mechanical properties. The crosslinked and mechanically stable porous hydrogels could be generated by simply mixing the two clickable Tetrazine-CS (TCS) and A-PEG-N components, generating a self-standing hydrogel within minutes. The leading candidate (TCS-8A-PEG-N (40 kD)), based on the mechanical and biocompatibility results, was further employed as a scaffold to improve wound closure and blood flow in vivo. The hydrogel demonstrated not only enhanced blood perfusion and an increased number of blood vessels, but also desirable fibrous matrix orientation and normal collagen deposition. Taken together, these results demonstrate the potential of the hydrogel to improve wound repair and hold promise for in situ skin tissue engineering applications.

Eosinophil plays a crucial role in intestinal mucositis induced by antineoplastic chemotherapy.

Mucositis is a major clinical complication associated with cancer treatment and may limit the benefit of chemotherapy. Leukocytes and inflammatory mediators have been extensively associated with mucositis severity. However, the role of eosinophils in the pathophysiology of chemotherapy-induced mucositis remains to be elucidated. Here, using GATA-1-deficient mice, we investigated the role of eosinophils in intestinal mucositis. There was marked accumulation of eosinophils in mice given irinotecan and eosinophil ablation inhibited intestinal mucositis. Treatment with Evasin-4, a chemokine receptor antagonist, reduced the recruitment of eosinophils and decreased irinotecan-induced mucositis. Importantly, Evasin-4 did not interfere negatively with the antitumour effects of irinotecan. Evasin-4 was of benefit for mice given high doses of irinotecan once Evasin-4-treated mice presented delayed mortality. Altogether, our findings suggest that Evasin-4 may have significant mucosal-protective effects in the context of antineoplastic chemotherapy and may, therefore, be useful in combination with anticancer treatment in cancer patients.

Retinoic acid-loaded solid lipid nanoparticles surrounded by chitosan film support diabetic wound healing in in vivo study.

Repair of tissue damaged in diabetic wounds is essential to minimize the cases of amputation of the limbs in millions of diabetic people around the world. Although the all-trans retinoic acid (ATRA) is described as a potential wound healing agent, however its effects are controversial due to adverse reactions that may impair the wound healing during the treatment schedules. Our aim was to design and characterize an ATRA-loaded solid lipid nanoparticles surrounded by chitosan film to promote an ATRA controlled release and to evaluate its effectiveness in promoting wound healing in a diabetic mouse model. The SLN-ATRA were developed using biocompatible lipids without using organic solvent. The SLN-ATRA had high drug entrapment efficiency (98.0 %) and low polydispersity index (PDI) and average diameter, respectively, 0.24 ± 0.02 and 83.0 ± 6 nm. The transmission electron microscope (TEM) image presented that the SLN-ATRA were homogeneous in size and had spherical structures. The incorporation of SLN-ATRA in the chitosan films propitiated a homogeneous distribution of the drug and a controlled drug release. Furthermore, in vivo assay proved that chitosan films containing SLN-ATRA accelerated the closure of wounds of diabetic mice when compared to the control chitosan films without ATRA. SLN-ATRA chitosan films also reduced leukocyte infiltrate in the wound bed, improved collagen deposition, and reduced scar tissue. No sign of skin irritation was observed. These results indicated that SLN-ATRA surrounded in chitosan films are a promising candidate to treat diabetic wounds, improving tissue healing.

P1G10, the Proteolytic Fraction from Vasconcellea cundinamarcensis, Stimulates Tissue Repair after Acute Exposure to Ultraviolet B Radiation.

BACKGROUND: P1G10 is a cysteine proteolytic fraction from Vasconcellea cundinamarcensis latex, obtained by chromatographic separation on Sephadex-G10 and ultrafiltration. This fraction enhances healing in different models of skin lesions, and displays a protective/healing effect against gastric ulcers, where it was suggested an antioxidant role. METHODS: We evaluated here the effect of topical treatment with P1G10, in mice lesions induced by UVB. RESULTS: After single exposure to 2.4 J cm-2 UVB, P1G10 reduced erythema, increased cellularity of hypodermis, enhanced MPO activity and IL1ß, and inhibited COX2 levels. These results point to an anti-inflammatory effect by P1G10. This fraction displayed antioxidant activity by reversing the depletion of glutathione (GSH), glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and reducing the catalase activity increased by UVB. These changes may be related to a reduction in MDA observed in groups treated with P1G10. P1G10 also inhibited MMP-9, caspase-3 and pkat while increasing p53 levels.

Angiotensin-(1-7) Promotes Resolution of Eosinophilic Inflammation in an Experimental Model of Asthma.

Defective apoptosis of eosinophils, the main leukocyte in the pathogenesis of asthma, and delay in its removal lead to lung damage and loss of pulmonary function due to failure in the resolution of inflammation. Here, we investigated the ability of angiotensin-(1-7) [Ang-(1-7)], a pivotal peptide of the renin-angiotensin system, to promote resolution of an allergic lung inflammatory response. Balb/c mice were sensitized and challenged with ovalbumin and treated with Ang-(1-7) at the peak of the inflammatory process. Bronchoalveolar lavage (BAL) fluid and lungs were collected 24 h after treatment. Different lung lobes were processed for histology to evaluate inflammatory cell infiltration, airway and pulmonary remodeling, total collagen staining, and measurements of (i) collagen I and III mRNA expression by qRT-PCR; (ii) ERK1/2, IκB-α, and GATA3 protein levels by Western blotting; and (iii) eosinophilic peroxidase activity. Total number of inflammatory cells, proportion of apoptotic eosinophils and immunofluorescence for caspase 3 and NF-κB in leukocytes were evaluated in the BAL. Mas receptor immunostaining was evaluated in mouse and human eosinophils. Engulfment of human polimorphonuclear cells by macrophages, efferocytosis, was evaluated in vivo. Ang-(1-7) reduced eosinophils in the lung and in the BAL, increased the number of apoptotic eosinophils, shown by histology criteria and by increase in caspase 3 immunostaining. Furthermore, Ang-(1-7) decreased NF-kB immunostaining in eosinophils, reduced GATA3, ERK1/2, and IκB-α expression in the lung and decreased pulmonary remodeling and collagen deposition. Importantly, Ang-(1-7) increased efferocytosis. Our results demonstrate, for the first time, Ang-(1-7) activates events that are crucial for resolution of the inflammatory process of asthma and promotion of the return of lung homeostasis, indicating Ang-(1-7) as novel endogenous inflammation-resolving mediator.

Chronic allergic pulmonary inflammation is aggravated in angiotensin-(1-7) Mas receptor knockout mice.

The angiotensin-(1-7) [ANG-(1-7)]/Mas receptor pathway is currently recognized as a counterbalancing mechanism of the renin-angiotensin system in different pathophysiological conditions. We have previously described that treatment with ANG-(1-7) attenuates lung inflammation and remodeling in an experimental model of asthma. In the present study, we investigated whether lack of the Mas receptor could alter the inflammatory response in a model of chronic allergic lung inflammation induced by ovalbumin (OVA). Mas receptor wild-type (MasWT) and knockout (MasKO) mice were subjected to four doses of OVA (20 µg/mice ip) with a 14-day interval. At the 21st day, nebulization with OVA (1%) was started, three times per week until the 46th day. Control groups received saline (0.9% ip) and were nebulized with saline (0.9%). MasWT-OVA developed a modest inflammatory response and minor pulmonary remodeling to OVA challenge. Strikingly, MasKO-OVA presented a significant increase in inflammatory cell infiltrate, increase in extracellular matrix deposition, increase in thickening of the alveolar parenchyma, increase in thickening of the smooth muscle layer of the pulmonary arterioles, increase in proinflammatory cytokine and chemokine levels in the lungs, characteristic of chronic asthma. Additionally, MasKO-OVA presented an increase in ERK1/2 phosphorylation compared with MasWT-OVA. Furthermore, MasKO-OVA showed a worse performance in a test of maximum physical exercise compared with MasWT-OVA. Our study shows that effects triggered by the Mas receptor are important to attenuate the inflammatory and remodeling processes in a model of allergic lung inflammation in mice. Our data indicate that impairment of the ANG-(1-7)/Mas receptor pathway may lead to worsening of the pathophysiological changes of asthma.

Skin wound healing is accelerated and scarless in the absence of commensal microbiota.

The commensal microbiota has a high impact on health and disease by modulating the development and homeostasis of host immune system. Immune cells are involved in virtually every aspect of the wound repair process; however, the impact of commensal microbiota on skin wound healing is largely unknown. In this study, we evaluated the influence of commensal microbiota on tissue repair of excisional skin wounds by using germ-free (GF) Swiss mice. We observed that macroscopic wound closure rate is accelerated in the absence of commensal microbiota. Accordantly, histologically assessed wound epithelization was accelerated in GF in comparison with conventional (CV) Swiss mice. The wounds of GF mice presented a significant decrease in neutrophil accumulation and an increase in mast cell and macrophage infiltration into wounds. Interestingly, alternatively activated healing macrophage-related genes were highly expressed in the wound tissue of GF mice. Moreover, levels of the anti-inflammatory cytokine IL-10, the angiogenic growth factor VEGF and angiogenesis were higher in the wound tissue of those mice. Conversely, scarring and levels of the profibrogenic factor TGF-ß1 were greatly reduced in GF mice wounded skin when compared with CV mice. Of note, conventionalization of GF mice with CV microbiota restored wound closure rate, neutrophil and macrophage accumulation, cytokine production, and scarring to the same extent as CV mice. Overall, our findings suggest that, in the absence of any contact with microbiota, skin wound healing is accelerated and scarless, partially because of reduced accumulation of neutrophils, increased accumulation of alternatively activated healing macrophages, and better angiogenesis at wound sites.

Plasmin induces in vivo monocyte recruitment through protease-activated receptor-1-, MEK/ERK-, and CCR2-mediated signaling.

The plasminogen (Plg)/plasmin (Pla) system is associated with a variety of biological activities beyond the classical dissolution of fibrin clots, including cell migration, tissue repair, and inflammation. Although the capacity of Plg/Pla to induce cell migration is well defined, the mechanism underlying this process in vivo is elusive. In this study, we show that Pla induces in vitro migration of murine fibroblasts and macrophages (RAW 264.7) dependent on the MEK/ERK pathway and by requiring its proteolytic activity and lysine binding sites. Plasmin injection into the pleural cavity of BALB/c mice induced a time-dependent influx of mononuclear cells that was associated with augmented ERK1/2 and IκB-α phosphorylation and increased levels of CCL2 and IL-6 in pleural exudates. The inhibition of protease activity by using a serine protease inhibitor leupeptin or two structurally different protease-activated receptor-1 antagonists (SCH79797 and RWJ56110) abolished Pla-induced mononuclear recruitment and ERK1/2 and IκB-α phosphorylation. Interestingly, inhibition of the MEK/ERK pathway abolished Pla-induced CCL2 upregulation and mononuclear cell influx. In agreement with a requirement for the CCL2/CCR2 axis to Pla-induced cell migration, the use of a CCR2 antagonist (RS504393) prevented the Plg/Pla-induced recruitment of mononuclear cells to the pleural cavity and migration of macrophages at transwell plates. Therefore, Pla-induced mononuclear cell recruitment in vivo was dependent on protease-activated receptor-1 activation of the MEK/ERK/NF-κB pathway, which led to the release of CCL2 and activation of CCR2.

Deletion of the chemokine receptor CCR2 attenuates foreign body reaction to implants in mice.

Subcutaneous implantation of synthetic materials and biomedical devices often induces abnormal tissue healing - the foreign body reaction - which impairs their function. Here we investigated the role of the chemokine receptor CCR2 in this reaction to subcutaneous implants in mice. We measured angiogenesis, inflammation and fibrogenesis induced by implantation, for 1, 4, 7 and 14days, of polyether-polyurethane sponges in mice with genetic deletion of CCR2 (KO) and WT mice. Blood flow was determined by dye diffusion and laser Doppler perfusion techniques. Cytokines (VEGF, TNF-α, CCL2, TGF-ß1) were measured by ELISA. Histochemical methods were used to assess collagen deposition and macrophage-derived giant cells in the implants. Skin and implant blood flow was lower in CCR2 KO than in WT mice, as were other aspects of neo-vascularization of the implants. Neutrophil accumulation was increased in KO implants but macrophage accumulation was decreased. Implant content of CCL2 was higher in KO implants, but TGF-ß1, collagen deposition and the number of foreign body giant cells were lower than in WT implants. Deletion of CCR2 decreased blood flow in normal skin and inhibited neo-vascularization, chronic inflammation and fibrogenesis in subcutaneous implants. The chemokine receptor CCR2 plays an important role in both normal skin and in the reaction elicited by subcutaneous implantation of a foreign body.

Brazilian green propolis modulates inflammation, angiogenesis and fibrogenesis in intraperitoneal implant in mice.

BACKGROUND: Chronic inflammatory processes in the peritoneal cavity develop as a result of ischemia, foreign body reaction, and trauma. Brazilian green propolis, a beeswax product, has been shown to exhibit multiple actions on inflammation and tissue repair. Our aim was to investigate the effects of this natural product on the inflammatory, angiogenic, and fibrogenic components of the peritoneal fibroproliferative tissue induced by a synthetic matrix. METHODS: Chronic inflammation was induced by placing polyether-polyurethane sponge discs in the abdominal cavity of anesthetized Swiss mice. Oral administration of propolis (500/mg/kg/day) by gavage started 24 hours after injury for four days. The effect of propolis on peritoneal permeability was evaluated through fluorescein diffusion rate 4 days post implantation. The effects of propolis on the inflammatory (myeloperoxidase and n-acetyl-ß-D-glucosaminidase activities and TNF-α levels), angiogenic (hemoglobin content-Hb), and fibrogenic (TGF-ß1 and collagen deposition) components of the fibrovascular tissue in the implants were determined 5 days after the injury. RESULTS: Propolis was able to decrease intraperitoneal permeability. The time taken for fluorescence to peak in the systemic circulation was 20±1 min in the treated group in contrast with 15±1 min in the control group. In addition, the treatment was shown to down-regulate angiogenesis (Hb content) and fibrosis by decreasing TGF-ß1 levels and collagen deposition in fibroproliferative tissue induced by the synthetic implants. Conversely, the treatment up-regulated inflammatory enzyme activities, TNF-α levels and gene expression of NOS2 and IFN-γ (23 and 7 fold, respectively), and of FIZZ1 and YM1 (8 and 2 fold) when compared with the untreated group. CONCLUSIONS: These observations show for the first time the effects of propolis modulating intraperitoneal inflammatory angiogenesis in mice and disclose important action mechanisms of the compound (downregulation of angiogenic components and activation of murine macrophage pathways).

Natriuretic peptide clearance receptor ligand (C-ANP4-23 ) attenuates angiogenesis in a murine sponge implant model.

Natriuretic peptide receptor-C activation by the synthetic ligand C-ANP-4-23, a specific agonist for this receptor, has been shown to inhibit key events of the angiogenic cascade, such as migration, proliferation and vascular endothelial growth factor (VEGF) production. In the present study we investigated whether C-ANP4-23 could also inhibit angiogenesis in the sponge model in vivo. To this end, we evaluated the effects of C-ANP4-23 on inflammatory and angiogenic components of the fibrovascular tissue induced by polyether polyurethane sponge implants in mice. Measurements of the haemoglobin content (µg/mg wet tissue) and blood flow (laser Doppler perfusion imaging) of the implants, used as an index of vascularization, revealed that single (200 ng) or multiple (200 ng/day, 5 days) doses of C-ANP4-23 reduced angiogenesis in the implants relative to the phosphate-buffered saline-treated group. The peptide exerted an inhibitory effect on nitric oxide production (nitrite levels) and had a dual effect on VEGF levels, depending on the number of doses (i.e. stimulation at 4 days after one dose; inhibition at 7 days after five doses). Histological analysis corroborated the biochemical and functional parameters indicative of inhibition of neovascularization (decreased vessel number) by C-ANP4-23 . The peptide failed to modulate inflammation in our system. The inhibitory effect of C-ANP4-23 on the angiogenic component of the fibrovascular tissue induced by the synthetic matrix extends the range of the its actions and may indicate its therapeutic potential in controlling angiogenesis in fibroproliferative diseases.

Blockade of cannabinoid receptors reduces inflammation, leukocyte accumulation and neovascularization in a model of sponge-induced inflammatory angiogenesis.

OBJECTIVE: Angiogenesis depends on a complex interaction between cellular networks and mediators. The endocannabinoid system and its receptors have been shown to play a role in models of inflammation. Here, we investigated whether blockade of cannabinoid receptors may interfere with inflammatory angiogenesis. MATERIALS AND METHODS: Polyester-polyurethane sponges were implanted in C57Bl/6j mice. Animals received doses (3 and 10 mg/kg/daily, s.c.) of the cannabinoid receptor antagonists SR141716A (CB1) or SR144528 (CB2). Implants were collected at days 7 and 14 for cytokines, hemoglobin, myeloperoxidase, and N-acetylglucosaminidase measurements, as indices of inflammation, angiogenesis, neutrophil and macrophage accumulation, respectively. Histological and morphometric analysis were also performed. RESULTS: Cannabinoid receptors expression in implants was detected from day 4 after implantation. Treatment with CB1 or CB2 receptor antagonists reduced cellular influx into sponges at days 7 and 14 after implantation, although CB1 receptor antagonist were more effective at blocking leukocyte accumulation. There was a reduction in TNF-α, VEGF, CXCL1/KC, CCL2/JE, and CCL3/MIP-1α levels, with increase in CCL5/RANTES. Both treatments reduced neovascularization. Dual blockade of cannabinoid receptors resulted in maximum inhibition of inflammatory angiogenesis. CONCLUSIONS: Blockade of cannabinoid receptors reduced leukocyte accumulation, inflammation and neovascularization, suggesting an important role of endocannabinoids in sponge-induced inflammatory angiogenesis both via CB1 and CB2 receptors.

Phosphoinositide 3-kinase γ plays a critical role in bleomycin-induced pulmonary inflammation and fibrosis in mice.

PI3Kγ is central in signaling diverse arrays of cellular functions and inflammation. Pulmonary fibrosis is associated with pulmonary inflammation, angiogenesis, and deposition of collagen and is modeled by instillation of bleomycin. The role of PI3Kγ in mediating bleomycin-induced pulmonary inflammation and fibrosis in mice and potential mechanisms involved was investigated here. WT or PI3Kγ KO mice were instilled with bleomycin and leukocyte subtype influx, cytokine and chemokine levels, and angiogenesis and tissue fibrosis evaluated. The activation of lung-derived leukocytes and fibroblasts was evaluated in vitro. The relevance of PI3Kγ for endothelial cell function was evaluated in HUVECs. PI3Kγ KO mice had greater survival and weight recovery and less fibrosis than WT mice after bleomycin instillation. This was associated with decreased production of TGF-ß(1) and CCL2 and increased production of IFN-γ and IL-10. There was reduced expression of collagen, fibronectin, α-SMA, and von Willebrand factor and decreased numbers and activation of leukocytes and phosphorylation of AKT and IκB-α. PI3Kγ KO mice had a reduced number and area of blood vessels in the lungs. In vitro, treatment of human endothelial cells with the PI3Kγ inhibitor AS605240 decreased proliferation, migration, and formation of capillary-like structures. AS605240 also decreased production of collagen by murine lung-derived fibroblasts. PI3Kγ deficiency confers protection against bleomycin-induced pulmonary injury, angiogenesis, and fibrosis through the modulation of leukocyte, fibroblast, and endothelial cell functions. Inhibitors of PI3Kγ may be beneficial for the treatment of pulmonary fibrosis.

Role of endothelin receptors in the control of central nervous system parasitism in Trypanosoma cruzi infection in rats.

Endothelin has been implicated in the pathogenesis of experimental and human Chagas disease. In the present study, we investigated whether the treatment with bosentan, an antagonist of both ET(A)/ET(B) endothelin receptors, modified parasite load and inflammation in the central nervous system (CNS) of Trypanosomacruzi-infected rats. The cerebellum was the most affected region in the CNS with marked parasitism and inflammation. Treatment with bosentan enhanced parasitemia and CNS parasitism, but control of infection was eventually attained. There was also an increase in the levels of the cytokines TNF-alpha, IL-10, IFN-gamma, CCL2/MCP-1, CCL3/MIP-1alpha and CCL5/RANTES in the brain of infected animals at days 9, 13 and 18 after infection. Overall, bosentan has some effects on the expression of certain cytokines and this may be related to the initial enhanced parasite load. Altogether, our data suggest that endothelin action via ET(A) and ET(B) receptors may play a role in the initial resistance of the CNS to T. cruzi infection in rats.

Role of the chemokines CCL3/MIP-1 alpha and CCL5/RANTES in sponge-induced inflammatory angiogenesis in mice.

OBJECTIVE: We examined the potential contribution of CCL3 and CCL5 to inflammatory angiogenesis in mice. METHODS: Polyester-polyurethane sponges were implanted in mice and blood vessel counting and hemoglobin, myeloperoxidase and N-acetylglucosaminidase measurements used as indexes for vascularization, neutrophil and macrophage accumulation, respectively. RESULTS: CCL3 and CCL5 were expressed throughout the observation period. Exogenous CCL3 enhanced angiogenesis in WT, but angiogenesis proceeded normally in CCL3(-/-) mice, suggesting that endogenous CCL3 is not critical for sponge-induced angiogenesis in mice. CCL5 expression was detected at day 1, but levels significantly increased thereafter. Exogenous CCL5 reduced angiogenesis in WT mice possible via CCR5 as CCL5 was without an effect in CCR5(-/-) mice. Treatment of WT with the CCR1/CCR5 antagonist, Met-RANTES, prevented neutrophil and macrophage accumulation, but enhanced sponge vascularization. CONCLUSION: Thus, endogenous CCL3 appears not to play a role in driving sponge-induced inflammatory angiogenesis in mice. The effects of CCL5 were anti-angiogenic and appeared to be mediated via activation of CCR5.

Human CD133+ progenitor cells promote the healing of diabetic ischemic ulcers by paracrine stimulation of angiogenesis and activation of Wnt signaling.

We evaluated the healing potential of human fetal aorta-derived CD133(+) progenitor cells and their conditioned medium (CD133(+) CCM) in a new model of ischemic diabetic ulcer. Streptozotocin-induced diabetic mice underwent bilateral limb ischemia and wounding. One wound was covered with collagen containing 2x10(4) CD133(+) or CD133(-) cells or vehicle. The contralateral wound, covered with only collagen, served as control. Fetal CD133(+) cells expressed high levels of wingless (Wnt) genes, which were downregulated following differentiation into CD133(-) cells along with upregulation of Wnt antagonists secreted frizzled-related protein (sFRP)-1, -3, and -4. CD133(+) cells accelerated wound closure as compared with CD133(-) or vehicle and promoted angiogenesis through stimulation of endothelial cell proliferation, migration, and survival by paracrine effects. CD133(+) cells secreted high levels of vascular endothelial growth factor (VEGF)-A and interleukin (IL)-8. Consistently, CD133(+) CCM accelerated wound closure and reparative angiogenesis, with this action abrogated by co-administering the Wnt antagonist sFRP-1 or neutralizing antibodies against VEGF-A or IL-8. In vitro, these effects were recapitulated following exposure of high-glucose-primed human umbilical vein endothelial cells to CD133(+) CCM, resulting in stimulation of migration, angiogenesis-like network formation and induction of Wnt expression. The promigratory and proangiogenic effect of CD133(+) CCM was blunted by sFRP-1, as well as antibodies against VEGF-A or IL-8. CD133(+) cells stimulate wound healing by paracrine mechanisms that activate Wnt signaling pathway in recipients. These preclinical findings open new perspectives for the cure of diabetic ulcers.

Activation of the PI3K/Akt pathway early during vaccinia and cowpox virus infections is required for both host survival and viral replication.

Viral manipulation of the transduction pathways associated with key cellular functions such as actin remodeling, microtubule stabilization, and survival may favor a productive viral infection. Here we show that consistent with the vaccinia virus (VACV) and cowpox virus (CPXV) requirement for cytoskeleton alterations early during the infection cycle, PBK/Akt was phosphorylated at S473 [Akt(S473-P)], a modification associated with the mammalian target of rapamycin complex 2 (mTORC2), which was paralleled by phosphorylation at T308 [Akt(T308-P)] by PI3K/PDK1, which is required for host survival. Notably, while VACV stimulated Akt(S473-P/T308-P) at early (1 h postinfection [p.i.]) and late (24 h p.i.) times during the infective cycle, CPXV stimulated Akt at early times only. Pharmacological and genetic inhibition of PI3K (LY294002) or Akt (Akt-X and a dominant-negative form of Akt-K179M) resulted in a significant decline in virus yield (from 80% to >/=90%). This decline was secondary to the inhibition of late viral gene expression, which in turn led to an arrest of virion morphogenesis at the immature-virion stage of the viral growth cycle. Furthermore, the cleavage of both caspase-3 and poly(ADP-ribose) polymerase and terminal deoxynucleotidyl transferase-mediated deoxyuridine nick end labeling assays confirmed that permissive, spontaneously immortalized cells such as A31 cells and mouse embryonic fibroblasts (MEFs) underwent apoptosis upon orthopoxvirus infection plus LY294002 treatment. Thus, in A31 cells and MEFs, early viral receptor-mediated signals transmitted via the PI3K/Akt pathway are required and precede the expression of viral antiapoptotic genes. Additionally, the inhibition of these signals resulted in the apoptosis of the infected cells and a significant decline in viral titers.