Vascular Recovery Research Articles

Stem cell compartmentalization in diabetes and high cardiovascular risk reveals the role of DPP-4 in diabetic stem cell mobilopathy

Bone marrow (BM) derived stem and progenitor cells contribute to cardiovascular homeostasis and are affected by cardiovascular risk factors. We devised a clinical data-driven approach to test candidate stem cell mobilizing mechanisms in pre-clinical models. We found that PB and BM CD34+ cell counts were directly correlated, and that most circulating CD34+ cells were viable, non-proliferating and derived from the BM. Thus, we analyzed PB and BM CD34+ cell levels as a two-compartment model in 72 patients with or without cardiovascular disease. Self-organizing maps showed that disturbed compartmentalization of CD34+ cells was associated with aging and cardiovascular risk factors especially diabetes. High activity of DPP-4, a regulator of the mobilizing chemokine SDF-1α, was associated with altered stem cell compartmentalization. For validation of these findings, we assessed the role of DPP-4 in the BM mobilization response of diabetic rats. Diabetes differentially affected DPP-4 activity in PB and BM and impaired stem/progenitor cell mobilization after ischemia or G-CSF administration. DPP-4 activity in the BM was required for the mobilizing effect of G-CSF, while in PB it blunted ischemia-induced mobilization. Indeed, DPP-4 deficiency restored ischemia (but not G-CSF)-induced stem cell mobilization and improved vascular recovery in diabetic animals. In conclusion, the analysis of stem cell compartmentalization in humans led us to discover mechanisms of BM unresponsiveness in diabetes determined by tissue-specific DPP-4 dysregulation.

Abstract 101: Endothelial-Specific Fibroblast Growth Factor Receptor 1 and 2 Deletion Impairs Vascular Remodeling and Recovery in an In Vivo Closed-Chest Model of Cardiac Ischemia-Reperfusion Injury

Introduction: Fibroblast growth factor (FGF) signaling is cardioprotective in various models of myocardial infarction. FGF receptors (FGFRs) are expressed in multiple cell types in the adult heart, but the cell type-specific FGFR signaling which mediates different cardioprotective endpoints is currently unknown. Hypothesis: We hypothesize that endothelial FGFR signaling is essential for vascular remodeling after cardiac ischemia-reperfusion (IR) injury. Methods: We have generated mice with a conditional ablation of the Fgfr1 and Fgfr2 genes in endothelial cells ( Tie2-Cre, Fgfr1 f/f , Fgfr2 f/f ). These mice and littermate controls were subjected to a clinically relevant, in vivo , closed-chest model of cardiac IR injury which includes 90 minutes of occlusion of the left anterior descending artery followed by reperfusion for 7 days. Echocardiography was performed at baseline, day 1, and day 7 of reperfusion, and histological staining was performed on day 7. Results: Tie2-Cre, Fgfr1 f/f , Fgfr2 f/f mice had no baseline abnormalities in cardiac morphometry or function. When subjected to closed-chest, regional cardiac IR injury, Tie2-Cre, Fgfr1 f/f , Fgfr2 f/f mice showed significantly increased infarct size compared to controls at 7 days (37±3% vs. 15±5%, p<0.05) but not 1 day after reperfusion. Tie2-Cre, Fgfr1 f/f , Fgfr2 f/f mice also showed significantly worsened cardiac function (ejection fraction, stroke volume, and fractional shortening) compared to controls at 7 days (p<0.05) but not 1 day after reperfusion. Myocyte cross-sectional area measurement showed no impairment in the cardiac hypertrophic response in the Tie2-Cre, Fgfr1 f/f , Fgfr2 f/f mice. Tie2-Cre, Fgfr1 f/f , Fgfr2 f/f mice have normal vessel density in the non-injured state, but after cardiac IR injury, Tie2-Cre, Fgfr1 f/f , Fgfr2 f/f hearts showed significantly decreased vessel density (both capillaries and smooth muscle actin containing vessels, p<0.05) compared to controls in the peri-infarct area. Conclusions: Ablation of FGFR1 and FGFR2 in endothelial cells results in impaired vascular remodeling, worsened cardiac functional recovery, and increased infarct size without affecting the cardiac hypertrophic response in an in vivo , closed-chest model of cardiac IR injury.

The attenuation of restenosis following arterial gene transfer using carbon nanotube coated stent incorporating TAT/DNAAng1+Vegf nanoparticles

This study report the development of a nanobiohybrid hydrogel based endovascular stent device capable of preventing postangioplasty in-stent restenosis (ISR) by promoting significant vascular endothelial recovery in a site-specific manner. The hydrogel is comprised of fibrin matrices, assembled layer-by-layer (LbL) on stent surface with alternate layers carrying endosomolytic Tat peptide/DNA nanoparticles (NPs) or NPs hybridized to polyacrylic acid (PAA) wrapped single-walled carbon nanotubes (NP–CNT). Here, the hydrogel works as a reservoir to carry, protect, and simultaneously deliver pro-angiogenic, vascular endothelial growth factor (Vegf) and Angiopoietin-1(Ang1), gene carrying NPs to the target site. In vitro results demonstrated that CNTs incorporated in the hydrogel layers play a major role in tuning the bioactivity of the stent. In addition, the developed stent formulation can significantly reduce the loss of therapeutics while traversing through the vessel and during deployment. In vivo experiments in balloon-injured canine femoral artery demonstrated that the NCS (+) group, carrying NPvegf+Ang1, can significantly enhance re-endothelialization of injured artery compared to control NCS (−), carrying NPNull, and bare metal stent (BMS) groups, attenuate stenosis (18.5±9.03% vs 39.56±13.8 vs 45.34±8.3%; n=8, p<0.05) and prevent neointima formation (1.53±0.36mm2 vs 2.51±0.27mm2 vs 2.66±0.14mm2; n=8, p<0.05) as analyzed angiography and histomorphometric analysis. These data collectively implicate that this new technology can be useful for stent and other biomedical devices through controlled delivery of multiple biotherapeutics.

Great toe transfer survival after thrombosis at 7 days

Sir, Survival of a great toe transfer depends on pedicle patency immediately postoperatively, as with all microvascular free flaps. Over time, neovascularisation from the recipient bed and surrounding wound margins into the graft may be sufficient to maintain flap viability independent of the primary vascular pedicle. It is not known how long the flow in the pedicle must persist for the flap to survive and there is no clear consensus regarding when free flaps become completely independent of their pedicle [1, 2]. The time it takes for a great toe transfer to develop its own blood supply from its recipient bed is still unknown. Our case shows that revascularisation can occur very early. We harvested a wrap-around flap from the great toe to reconstruct an amputated left thumb at the base of the proximal phalanx of a 32-year-old male smoker. The distal phalanx was harvested including the nail, most of the pulp, and the dorsal, lateral, and plantar skin of the great toe. An iliac crest bone graft was interposed between the proximal and distal ‘vascularised bone’ segments of the reconstructed thumb. The first dorsal metatarsal artery and accompanying vein were anastomosed at the ‘anatomical snuff box’. Total ischemic time was 2 h. Postoperatively, the patient received low molecular weight dextran (500 ml for 5 days) and aspirin (300 mg for 7 days). The patient was discharged on day 5—thanks to a perfect capillary refill of the reconstructed thumb. On day 7, clinical examination revealed a sluggish capillary refill, no bleeding from pin pricks on the tip of the reconstructed thumb and no Doppler signal at the microvascular anastomosis level. The patient returned to the operating room for re-exploration which showed complete thrombosis of the vascular pedicle (Fig. 1). All attempts at reanastomosis failed due to the extensive thrombosis throughout the vascular system of the flap. The patient was hospitalized and received intravenous heparin and dextran. He was discharged 2 days later without signs of vascular recovery of the thumb. A secondary surgical procedure was then scheduled. Surprisingly, 1 week later, the reconstructed thumb showed a normal capillary refill on the pulp giving us a reason to believe that the thumb was viable despite the present superficial epidermolysis, a small area of necrosis at the distal tip and still no detectable arterial Doppler signal distal to the anastomosis. After 10 months, the transplanted thumb showed satisfactory results (Figs. 2 and 3). Several authors have described clinical cases of free flap survival despite early loss of the pedicle blood supply [3, 4]. These papers suggest that after 12 days, adequate revascularisation from the recipient bed may allow at least partial flap survival. Recent clinical cases even suggest that the time may be further reduced [2, 5, 6] to 6–9 days. However, many authors have described complete loss of free flaps several months after surgery, thus highlighting the dependency of free flaps on their vascular pedicle up to 1 year after free tissue transfer [7, 8]. The process of revascularisation is believed to be dependent upon a variety of factors including type of flap used, flap thickness, quality of recipient site and ischemic time during transfer [2–4]. Free flaps are more likely to survive after vascular thrombosis if they have a sufficient marginal inset of their cutaneous portion and contact with the recipient bed [9]. It is evident, therefore, that the process of neovascularisation varies [2]. As already reported, the current consensus is that a free flap can survive after early interruption of the vascular pedicle. However, the cases reported describe the use of fasciocutaneous [1–5] or muscle free flaps [6], whereas in our case, we transferred a composite osteo-onychocutaneous free flap. Transplanted toes, similar to replanted thumbs, can survive even L. Lazzaro Hand Surgery Department, Azienda Ospedaliera Universitaria Integrata Verona, Verona, Italy

Regulation of Connexin43 Gap Junction Protein Triggers Vascular Recovery and Healing in Human Ocular Persistent Epithelial Defect Wounds

Transiently blocking the expression of the gap junction protein connexin43 using antisense oligodeoxynucleotides or blocking hemichannels with connexin mimetic peptides has been shown to significantly improve outcomes in a range of acute wound models. Less is known about their likely effects in nonhealing wounds. In the eye, prolonged inflammation and lack of epithelial recovery in nonhealing corneal epithelial wounds may lead to corneal opacity, blindness or enucleation. We report here the first human applications of antisense oligodeoxynucleotides that transiently block translation of connexin43 in a prospective study of five eyes with severe ocular surface burns (persistent epithelial defects), which were unresponsive to established therapy for 7days to 8weeks prior to treatment. Connexin43-specific antisense oligodeoxynucleotide was delivered in cold, thermoreversible Poloxamer407 gel under either an amniotic membrane graft or a bandage contact lens. The connexin43-specific antisense application reduced inflammation within 1-2days, and in all five eyes complete and stable corneal reepithelialization was obtained. Recovery of the vascular bed and limbal reperfusion appeared to precede corneal epithelial recovery. We conclude that connexin modulation provides a number of benefits for nonhealing ocular burn wounds, one of which is to promote vascular recovery.

Growth and regression of vasculature in healthy and diabetic mice after hindlimb ischemia

The formation of vascular networks during embryogenesis and early stages of development encompasses complex and tightly regulated growth of blood vessels, followed by maturation of some vessels, and spatially controlled disconnection and pruning of others. The adult vasculature, while more quiescent, is also capable of adapting to changing physiological conditions by remodeling blood vessels. Numerous studies have focused on understanding key factors that drive vessel growth in the adult in response to ischemic injury. However, little is known about the extent of vessel rarefaction and its potential contribution to the final outcome of vascular recovery. We addressed this topic by characterizing the endogenous phases of vascular repair in a mouse model of hindlimb ischemia. We showed that this process is biphasic. It encompasses an initial rapid phase of vessel growth, followed by a later phase of vessel rarefaction. In healthy mice, this process resulted in partial recovery of perfusion and completely restored the ability of mice to run voluntarily. Given that the ability to revascularize can be compromised by a cardiovascular risk factor such as diabetes, we also examined vascular repair in diabetic mice. We found that paradoxically both the initial growth and subsequent regression of collateral vessels were more pronounced in the setting of diabetes and resulted in impaired recovery of perfusion and impaired functional status. In conclusion, our findings demonstrate that the formation of functional collateral vessels in the hindlimb requires vessel growth and subsequent vessel rarefaction. In the setting of diabetes, the physiological defect was not in the initial formation of vessels but rather in the inability to sustain newly formed vessels.

In This Issue

HomeCirculation ResearchVol. 110, No. 8In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Originally published13 Apr 2012https://doi.org/10.1161/RES.0b013e3182571a16Circulation Research. 2012;110:1035Lack of Neutrophil-Derived CRAMP Reduces Atherosclerosis in Mice (p 1052)Döring et al reveal how neutrophils promote atherosclerosis, and suggest a way to stop it.Download figureDownload PowerPointAtherosclerosis is a chronic inflammatory disorder associated with lipid accumulation in the vessel wall. Although the inflammation is known to be promoted, at least in part, by neutrophils, the precise mechanism by which these cells contribute to atherogenesis remains unclear. When activated, neutrophils release secretory vesicles called granules, and certain granule proteins, such as CRAMP (or LL37 in humans), are able to recruit other inflammatory cell types. To determine whether this happens during atherosclerotic lesion formation, Döring et al examined atherosclerosis-prone mice lacking CRAMP. Sure enough, these mice had fewer inflammatory cells adhering to their blood vessel walls, which resulted in smaller atherosclerotic plaques containing a reduced proportion of macrophages. CRAMP previously has been detected in endothelial cells and macrophages, but the team showed that in atherosclerotic vessels, CRAMP was specifically upregulated in neutrophils. The team also found that CRAMP promotes inflammatory cell recruitment by interacting with their formyl-peptide membrane receptors. Blocking these receptors, or indeed CRAMP activity, may be an avenue toward atherosclerosis therapies, as suggested by the authors.Sensing Gene-Regulatory Hyperglycemic Changes by Set7 in Vascular Endothelial Cells (p 1067)Histone modifier, Set7, activates proinflammatory genes in response to high blood glucose, reportOkabe et al.Download figureDownload PowerPointHistone proteins in chromatin can be posttranslationally modified in a number of different ways, and these modifications can be indicative of particular states of gene expression. For example, the addition of a methyl group to lysine 4 of histone H3 is associated with gene activation. Okabe et al were studying the activation of proinflammatory genes in response to hyperglycemia and wondered if such H3 methylation might be involved. They examined Set7, an H3-methylating enzyme, and found that when human epithelial cells were exposed to high glucose, the enzyme relocated to the nucleus. There, Set7 activated two proinflammatory genes, IL-8 and HMOX1. However, only IL-8 displayed H3 methylation, suggesting Set7 can activate genes independent of its methylation activity. In mice subjected to transient hyperglycemia, the same gene activation and histone methylation were observed. And, interestingly, the histone modification lasted at least 1 week. This persistence, say the authors, might explain “hyperglycemic memory,” the observation that even transient spikes in blood glucose levels can activate pathophysiological pathways leading to diabetes and atherosclerosis.Revascularization of Ischemic Skeletal Muscle by Estrogen-Related Receptor-γ (p 1087)Transcription factor ERRγ prompts new blood vessel growth in ischemic muscle, report Matsakas et al.Download figureDownload PowerPointSkeletal muscle ischemia is a common disorder that often accompanies obesity, diabetes, atherosclerosis, and other cardiovascular and metabolic diseases. In severe cases, it can lead to tissue death and limb amputation. Besides adopting a healthier lifestyle, undergoing vascular surgery is among the few available treatment options. Thus, noninvasive treatments are desperately needed. Matsakas et al now suggest that boosting muscle ERRγ levels might be one such noninvasive approach. ERRγ is known to activate a network of proangiogenic and oxidative metabolism factors in mouse skeletal muscle, but whether it could actually promote vascular recovery was unknown. To find out, the team occluded blood vessels in the hind limbs of mice to induce muscle ischemia. Within 1 or 2 weeks, mice that overexpressed ERRγ showed improved revascularization, blood perfusion, and reversal of myofiber damage compared with their wild-type counterparts. The transgenic mice also displayed higher expression levels of proangiogenic factors in the ischemic muscle. The authors suggest that boosting ERRγ levels might be a potential therapy not only for ischemia of the muscle but also for that of the heart or brain.Written by Ruth Williams. Previous Back to top Next FiguresReferencesRelatedDetails April 13, 2012Vol 110, Issue 8 Advertisement Article InformationMetrics © 2012 American Heart Association, Inc.https://doi.org/10.1161/RES.0b013e3182571a16 Originally publishedApril 13, 2012 PDF download Advertisement

Revascularization of Ischemic Skeletal Muscle by Estrogen-Related Receptor-γ

Oxidative myofibers in the skeletal muscles express high levels of angiogenic factors, have dense vasculature, and promptly revascularize during ischemia. Estrogen-related receptor-gamma (ERRγ) activates genes that govern metabolic and vascular features typical to oxidative myofibers. Therefore, ERRγ-dependent remodeling of the myofibers may promote neoangiogenesis and restoration of blood perfusion in skeletal muscle ischemia. To investigate the muscle fiber type remodeling by ERRγ and its role in the vascular recovery of ischemic muscle. Using immunohistology, we show that skeletal muscle-specific transgenic overexpression of ERRγ increases the proportions of oxidative and densely vascularized type IIA and IIX myofibers and decreases glycolytic and less vascularized type IIB myofibers. This myofiber remodeling results in a higher basal blood flow in the transgenic skeletal muscle. By applying unilateral hind limb ischemia to transgenic and wild-type mice, we found accelerated revascularization (fluorescent microangiography), restoration of blood perfusion (laser Doppler flowmetry), and muscle repair (Evans blue dye exclusion) in transgenic compared to wild-type ischemic muscles. This ameliorative effect is linked to enhanced neoangiogenesis (CD31 staining and microfil perfusion) by ERRγ. Using cultured muscle cells in which ERRγ is inactivated, we show that the receptor is dispensable for the classical hypoxic response of transcriptional upregulation and secretion of vascular endothelial growth factor A. Rather, the ameliorative effect of ERRγ is linked to the receptor-mediated increase in oxidative myofibers that inherently express and secrete high levels of angiogenic factors. The ERRγ is a hypoxia-independent inducer of neoangiogenesis that can promote reparative revascularization.

The application of tissue engineering in reversing mitomycin C‐induced ischemic conjunctiva

The purpose of this study is to evaluate the angiogenic potential of collagen-glycosaminoglycan (CG) matrices in mitomycin C-induced ischemic conjunctival defect, in New Zealand white rabbits. After creating a conjunctival defect at the center of ischemic conjunctiva, a CG matrix was implanted into subconjunctival space to evaluate the conjunctival reepithelialization and angiogenesis during the wound healing process. In the grafted group, the vessel count of the healed conjunctiva was substantially elevated by two fold within the initial 4 weeks and the increased vascular content originated mostly from the fornix site. The rate of conjunctival reepithelialization was not retarded in the grafted group, and the final thickness of healed conjunctiva was similar in both grafted and ungrafted groups. The histological studies revealed that the collagen matrix did not elicit pronounced inflammatory reaction and the regenerated conjunctiva showed loosely arranged collagen deposition without significant scar formation. The α SMA staining positive myofibroblasts were identified in the acute inflammatory stage and were absent, 8 weeks after implantation in both groups. The results indicated that the porous collagen scaffold was able to enhance vascularization and physiological recovery of ischemic conjunctival defect, implying a potential alternative therapy for the ischemic leaking bleb after glaucoma filtrating surgery in ophthalmic practices.

Recognition of changes in microvascular and microstructural patterns upon magnifying endoscopy predicted the presence of extranodal gastric MALToma

BACKGROUND AND OBJECTIVES: Gastric MALToma is difficult to recognize upon endoscopy. The aim of this study is to evaluate the application of microstructural and microvascular patterns in recognizing gastric MALToma on magnifying endoscopy. METHOD: All patients with diagnosis of gastric MALToma upon histology were recruited. They received magnifying endoscopy to observe for changes in microstructural and microvascular patterns. For patients with H pylori, eradication therapy would be given. For those without, appropriate treatments including gastrectomy or chemotherapy were commenced accordingly. Patients treated with H pylori eradication and non-operative treatments received follow-up magnifying endoscopy, and the same features were observed to predict the response to these treatments. RESULTS: From 2004 to 2007, nine patients presented to with epigastric pain, dyspepsia and belching. All patients were confirmed to have MALToma upon initial biopsy. Five patients had H pylori infection and received eradication. Two patients without H pylori were treated with Laparoscopic total gastrectomy. Two patients had pulmonary metastasis and treated with chemotherapy. Under magnifying endoscopy, all the lesions demonstrated either absence or irregular gastric pits. Moreover, there was consistently appearance of spider-shaped vascular pattern. Five patients with H pylori eradication had follow-up magnifying endoscopy, four of them showed resolution of abnormal vascular pattern and recovery of gastric pits. CONCLUSION: Abnormal spider like vasculature and disappearance of gastric pits are diagnostic features upon magnifying endoscopy for gastric MALToma. These features enhanced the diagnosis and assessment of extent of involvement during primary endoscopy, as well as follow-up surveillance for response to non-operative treatments.

Abstract P039: Encapsulation of Human Mesenchymal Stem Cells in Alginate Microspheres to Enhance Paracrine-Mediated Vascular Recovery

Bone marrow cells migrate to ischemic areas and facilitate revascularization mostly through paracrine mechanisms. As a result, therapeutic strategies for vascular repair have focused on increasing the concentration of these cells at the site of ischemia by direct injection. These strategies have led to encouraging results in clinical trials, yet the number of cells that remain at the site of delivery is very low. The goal of this study is to design an effective cell delivery system to enhance revascularization by maintaining large numbers of cells at the ischemic site, thus increasing the local delivery of angiogenic factors. To achieve this goal, we encapsulated bone marrow derived human mesenchymal stem cells (hMSCs) in non-degradable, barium-gelled alginate spheres prior to delivery to the ischemic site. Our microspheres are biocompatible, do not generate a fibrotic response, and are stable for extended periods of time. They prevent incorporation of cells into the neighboring tissue and protect implanted cells from immune mediated clearance or wash out by the host capillary and lymphatic systems. The microspheres allow diffusion of molecules as large as 80 kDa (which includes most angiogenic factors) but not of larger molecules such as IgG. To test this cell delivery system in vivo, we used a model of murine hind limb ischemia in athymic nude mice. We delivered encapsulated hMSCs, or, as a control, empty capsules, to the ischemic muscle. At various time points, we retrieved the capsules by gentle scooping, then assessed the viability of the encapsulated cells and their protein secretion profile. Approximately 70% of the cells remained viable within the first week and 10-20% within the first month after implantation. In addition, encapsulated cells secreted angiogenic factors (VEGF, bFGF, HGF, angiogenin) in vitro and in vivo. We also assessed recovery of perfusion in the hind limb and found that delivery of encapsulated cells enhanced vascular recovery as compared to empty capsules. The results obtained from this study show that microcapsules are an effective delivery vehicle of therapeutic cells for vascular repair. Encapsulation represents a powerful and highly translatable tool to enhance cell-based therapies in cardiovascular disease.

Can a Strychnos species be used as antiulcer agent? Ulcer healing action from alkaloid fraction of Strychnos pseudoquina St. Hil. (Loganiaceae)

Ethnopharmacological relevanceStrychnos pseudoquina St. Hil. (Loganiaceae) is one Brazilian native medicinal species described in the first edition of the Brazilian Official Pharmacopoeia in 1929. This medicinal plant, popularly known as “quina-quina”, “quina-branca” or “casca aromatica was very commonly used in folk medicine in tea form obtained from the bark and/or leaves as tonic, antipyretic, antimalarial and mainly against diseases of the liver, spleen and stomach. Aim of the studyPrevious study already characterized the gastroprotective action of this species The aim of the present study is to elucidate the mechanism of the healing process mediated by the methanolic extract (ME) and their enriched alkaloid fraction (EAF) from Strychnos pseudoquina in chronic gastric ulceration induced by 5% acetic acid in rats, an experimental model that accurately reflects human gastrointestinal disease. Material and methodsThe ME and EAF was administered orally in a single dose (based on previously study of dose–response curve) for 14 days after chronic ulceration was induced in rats. The healing effect of ME and EAF was evaluated by macroscopic and morphometric analyses, immunohistochemical assay (PCNA and SOD) and anti-Helicobacter pylori effect was evaluated by in vitro assay. ResultsOur results demonstrated that EAF significantly reduced border internal (42%) and external (38%) lesion area (mm2) by macroscopic analyses (P<0.05). Animals treated with EAF stimulated some proliferative factors by increasing the height of epithelial regenerative area and the expression of PCNA-positive nuclei. The number of vessels in gastric mucosa of rats treated with EAF reveals an expressive increase (4 times more than vehicle treatment) of vessels that stimulate cells proliferation in the healing region. These results suggest that the recovery of vascularization of the ulcerated area is involved in the healing action of alkaloid fraction of Strychnos pseudoquina. The MIC (minimum inhibitory concentration) of 75μg/ml from EAF showed an effective in vitro anti-Helicobacter pylori action of this fraction. EAF also was quite effective in the process of SOD release that is an important protective factor against bacterial agents. The efficacy of EAF was accomplished safely without presenting any alteration of toxicological parameters during 14 day of treatment. ConclusionsThe expressive gastric healing effect by increasing cellular proliferation together with expression of SOD activity and antibacterial action against Helicobacter pylori confirm the efficacy of this species in heal gastric mucosa and these results are a important contribution to the knowledge of a crude drug presents at the Brazilian Official Pharmacopoeia since 1929.

Endothelial Progenitor Cells: A Novel Tool for the Therapy of Ischemic Diseases

Circulating endothelial progenitor cells (EPCs) are believed to home to sites of neovascularization, contributing to vascular regeneration either directly via incorporation into newly forming vascular structures or indirectly via the secretion of pro-angiogenic growth factors, thereby enhancing the overall vascular and hemodynamic recovery of ischemic tissues. The therapeutic application of EPCs has been shown to be effective in animal models of ischemia, and we as well as other groups involved in clinical trials have demonstrated that the use of EPCs was safe and feasible for the treatment of critical limb ischemia and cardiovascular diseases. However, many issues in the field of EPC biology, especially in regard to the proper and unambiguous molecular characterization of these cells, still remain unresolved, hampering not only basic research but also the effective therapeutic use and widespread application of these cells. Further, recent evidence suggests that several diseases and pathological conditions are correlated with a reduction in the number and biological activity of EPCs, making the development of novel strategies to overcome the current limitations and shortcomings of this promising but still limited therapeutic tool by refinement and improvement of EPC purification, expansion, and administration techniques, a rather pressing issue.

Impaired hemodynamic response to meal intake in insulin-resistant subjects: an impedance cardiography approach

In the postprandial state, insulin regulates metabolic and cardiovascular responses. In insulin resistance, the insulin action is impaired at both levels. However, postprandial hemodynamic responses are poorly characterized in this setting. We investigated fasting and postprandial cardiac and vascular hemodynamic responses in subjects with and without insulin resistance. Sixty-six atherosclerosis-free, healthy volunteers were studied in a fasted state and ≤180 min after ingestion of a mixed meal. The insulin sensitivity index was determined by using a minimal model analysis; hemodynamic response was monitored by using continuous impedance cardiography that allowed a reliable beat-to-beat noninvasive evaluation of stroke volume, cardiac contractility, and several derived variables. Subjects were divided into insulin-resistant (IR; n = 33) and insulin-sensitive (IS; n = 33) groups. After fasting, IR subjects had significantly higher values of systolic and diastolic blood pressures and the systemic vascular resistance index (SVRI) than did IS subjects. In the postprandial state, acute vasodilatation was comparable and synchronous (at 30 min) in IR and IS subjects (P = 0.209), but subsequent vascular tone recovery (30-180 min) was significantly impaired in IR subjects (P = 0.018), even after adjustment for age and sex (P = 0.031). Hemodynamic dysregulation was directly correlated with metabolic disturbances in the postprandial state. In basal and postprandial states, hemodynamic variables related to cardiac function were not significantly different in IR and IS subjects. IR subjects had a worse fasting vascular performance than did IS subjects. In the postprandial phase, insulin resistance was associated with a shorter duration of vasodilatation in the absence of an altered cardiac performance. Peripheral hemodynamic alterations in fasting and postprandial states may have a negative effect on cardiovascular performance in IR patients.

Abstract 3600: The prominent safety profile of TAS-115, a novel c-Met + VEGFR dual kinase inhibitor can lead to potentiate the efficacy via durable inhibition of angiogenesis

Abstract c-Met is a receptor tyrosine kinase involved in various malignant phenotypes of diverse cancers. The inhibition of VEGF/VEGFR has been established as a standard therapy for several cancers. Therefore, their dual inhibition is considered to be one of most attractive strategy for cancer therapy. However, unexpectedly success of VEGFR inhibitors (VEGFRI) has been concurrently associated with emerging new type toxicities which result in occasional patient's death and requiring drug off period, frequent interruptions or dose reduction. Moreover, those interruptions resulted in a rapid tumor regrowth due to the aggressive angiogenic signaling which can confer the drug resistance to VEGFRI. These issues are considered to be main reason for the lack of success of their combination with other chemotherapeutics and the limitation of their usage. This sequence of events prompted us to develop a novel c-Met + VEGFRs dual inhibitor TAS-115, which has a high potency and prominent safety profile. A 4wks tolerability study in mice revealed no animal death even at more than 28 times higher drug exposure over the effective doses (ED50). Whereas, therapeutic index (MTD/ED50) of other VEGFRIs including foretinib, sunitinib and sorafenib were only 2-4. Similar safety profile was observed in the tox study in rats treated for 3wks, its therapeutic index was more than 10, while those of other VEGFRIs were only 0.6-2.5 due to severe toxicities. To confirm whether its better safety profile provide more benefit, longer term efficacy studies were conducted. There was no difference in efficacy within the first 2wks, however, over 2wks the animals in foretinib treatment group at MTD began to die and administration could not be continued. In contrast, TAS-115 dosing has continued for over 6wks without any toxic manifestation. The deference in their efficacies at MTD for over 4wks became greater (p&amp;lt;0.001). Moreover, to evaluate how the drug off period of VEGFRIs affect angiogenesis, using sunitinib as a reference, the efficacy study was done with 2wks on / 1wk off dosing regimen, mimicking its clinical setting. Although tumor growth was inhibited with 2wks consecutive dosing of sunitinib via angiogenesis inhibition, the 1wk rest caused a regrowth with recovery of vascularity. And even though its next 2wks dosing re-inhibited tumor growth, repeating these cycles allowed progressive growth with aggressive angiogenic phenotype including higher gene expressions of various angiogenic factors. Meanwhile, TAS-115 could completely inhibit tumor growth for more than 6wks via durable inhibition of angiogenesis. In addition, TAS-115 prolonged the life span in the peritoneal dissemination mouse models of human gastric cancer cell line resistant to sunitinib. Taken together, the prominent safety profile of TAS-115 can lead to more therapeutic benefit via a durable inhibition of angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3600. doi:10.1158/1538-7445.AM2011-3600

Transplanted Stem Cell-Secreted Vascular Endothelial Growth Factor Effects Poststroke Recovery, Inflammation, and Vascular Repair

Abstract Cell transplantation offers a novel therapeutic strategy for stroke; however, how transplanted cells function in vivo is poorly understood. We show for the first time that after subacute transplantation into the ischemic brain of human central nervous system stem cells grown as neurospheres (hCNS-SCns), the stem cell-secreted factor, human vascular endothelial growth factor (hVEGF), is necessary for cell-induced functional recovery. We correlate this functional recovery to hVEGF-induced effects on the host brain including multiple facets of vascular repair and its unexpected suppression of the inflammatory response. We found that transplanted hCNS-SCns affected multiple parameters in the brain with different kinetics: early improvement in blood-brain barrier integrity and suppression of inflammation was followed by a delayed spatiotemporal regulated increase in neovascularization. These events coincided with a bimodal pattern of functional recovery, with, an early recovery independent of neovascularization, and a delayed hVEGF-dependent recovery coincident with neovascularization. Therefore, cell transplantation therapy offers an exciting multimodal strategy for brain repair in stroke and potentially other disorders with a vascular or inflammatory component.

Postacute Ischemia Vascular Endothelial Growth Factor Transfer by Transferrin-Targeted Liposomes Attenuates Ischemic Brain Injury After Experimental Stroke in Rats

Our objective was to achieve the enhanced delivery of vascular endothelial growth factor (VEGF) to ischemically disordered brain through transferrin-coupled liposomes (Tf-PLs) via intravenous administration, and to observe the effect of Tf-VEGF-PLs on ischemic brain neuroprotection and angiogenesis. Cerebral VEGF overexpression was achieved with Tf-PLs by intravenous injection 48 hr after an acute stroke. β-Galactosidase expression was monitored; saline was injected as a control. The success of postischemic gene transduction was confirmed by β-galactosidase staining and by increased VEGF mRNA and protein in ischemic brain. Vascular density, neurological recovery, and ischemic area calculation were performed to evaluate the effect of Tf-VEGF-PLs. The positive expression of β-galactosidase indirectly indicated that VEGF was successfully delivered into brain by Tf-VEGF-PLs. VEGF mRNA in the Tf-VEGF-PL group 24 hr after injection was significantly higher than in the control group (p < 0.05). Western blot analysis showed that postischemic Tf-VEGF-PLs resulted in increased VEGF protein levels compared with VEGF-PLs and saline-administered rats (p < 0.05) 48 hr after administration. At 21 days after drug injection, we observed a significant decrease in infarct volume and better neurological function in the Tf-VEGF-PL-treated group, compared with the VEGF-PL group. FITC-dextran marking showed increased vascular density in the penumbra of Tf-VEGF-PL-treated hemispheres (245,873.9, number of microvessels per field) compared with that in VEGF-PL-treated hemispheres (139,801.3) or saline-treated hemispheres (102,175.5) (p < 0.05). The remainder of the cerebral blood flow after ischemia in the Tf-VEGF-PL group was significantly more than in the control groups (0.35 vs. 0.29, 0.21; p < 0.05). We conclude that the VEGF gene can be delivered noninvasively into the brain by Tf-VEGF-PLs. Postischemic treatment with Tf-VEGF-PLs effectively promoted neuroprotection and vascular regeneration in the chronic stage of cerebral infarction.

Primary Monocytes Regulate Endothelial Cell Survival Through Secretion of Angiopoietin-1 and Activation of Endothelial Tie2

Monocyte recruitment and interaction with the endothelium is imperative to vascular recovery. Tie2 plays a key role in endothelial health and vascular remodeling. We studied monocyte-mediated Tie2/angiopoietin signaling following interaction of primary monocytes with endothelial cells and its role in endothelial cell survival. The direct interaction of primary monocytes with subconfluent endothelial cells resulted in transient secretion of angiopoietin-1 from monocytes and the activation of endothelial Tie2. This effect was abolished by preactivation of monocytes with tumor necrosis factor-α. Although primary monocytes contained high levels of both angiopoietin 1 and 2, endothelial cells contained primarily angiopoietin 2. Seeding of monocytes on serum-starved endothelial cells reduced caspase-3 activity by 46 ± 5.1%, and 52 ± 5.8% after tumor necrosis factor-α treatment and decreased detected single-stranded DNA levels by 41 ± 4.2% and 40 ± 3.5%, respectively. This protective effect of monocytes on endothelial cells was reversed by Tie2 silencing with specific short interfering RNA. The antiapoptotic effect of monocytes was further supported by the activation of cell survival signaling pathways involving phosphatidylinositol 3-kinase, STAT3, and AKT. Monocytes and endothelial cells form a unique Tie2/angiopoietin-1 signaling system that affects endothelial cell survival and may play critical a role in vascular remodeling and homeostasis.

Contact and contactless diffuse reflectance spectroscopy: potential for recovery monitoring of vascular lesions after intense pulsed light treatment

Optical fiber contact probe diffuse reflectance spectroscopy and remote multispectral imaging methods in the spectral range of 400 to 1100 nm were used for skin vascular malformation assessment and recovery tracing after treatment by intense pulsed light. The results confirmed that oxy-hemoglobin relative changes and the optical density difference between lesion and healthy skin in the spectral region 500 to 600 nm may be successfully used for objective appraisal of the therapy effect. Color redness parameter a* = 2 is suggested as a diagnostic border to distinguish healthy skin and vascular lesions, and as the indicator of phototreatment efficiency. Valuable diagnostic information on large area (>5 mm) lesions and lesions with uncertain borders can be proved by the multispectral imaging method.

Activating Notch signaling post-SCI modulates angiogenesis in penumbral vascular beds but does not improve hindlimb locomotor recovery

Manipulation of Notch signaling has led to significant tumor shrinkage as well as recovery from several traumatic and ischemic injury models indicating its potential clinical application. We have tested both an agonist and antagonist of Notch signaling to study the effects of Notch-mediated angiogenesis on spinal cord vascular pathology following traumatic injury. Initial neonatal retinal vascularization assays showed their respective bioactivities in vivo. Mice were treated with either the antagonist Jagged1-Fc chimera (Jag1-Fc) or agonist Notch1 antibody (N1 Ab) immediately following a mid-thoracic contusive injury through an initial jugular bolus and tail vein injections for 3 days post-injury. After 14 days, activating Notch signaling decreased the overall vascular density within the penumbral gray matter compared to controls while maintaining the density of perfused vessels. Inhibiting Notch signaling did not change the density or perfusion of microvessels within the lesion penumbra. Furthermore, neither activation nor inhibition of Notch signaling significantly altered inflammation, hypoxia, and lesion volume in the epicenter and penumbra. Importantly, neither treatment changed locomotor function. In postnatal retinal vascular assays, administration of Jag1-Fc and N1 Ab increased and decreased both tip cell numbers and branch points in each treatment, respectively. However, these agents did not modulate primary CNS EC proliferation in vitro in spite of sufficient Notch ligand expression. We conclude that Notch signaling, while an important part of developmental angiogenesis, may play a lesser role in mediating vascular recovery following traumatic injury to the CNS.