Structure and replication cycle of a virus infecting climate-modulating alga Emiliania huxleyi.

The globally distributed marine alga Emiliania huxleyi has cooling effect on the Earth's climate. The population density of E. huxleyi is restricted by Nucleocytoviricota viruses, including E. huxleyi virus 201 (EhV-201). Despite the impact of E. huxleyi viruses on the climate, there is limited information about their structure and replication. Here, we show that the dsDNA genome inside the EhV-201 virion is protected by an inner membrane, capsid, and outer membrane. EhV-201 virions infect E. huxleyi by using fivefold vertices to bind to and fuse the virus' inner membrane with the cell plasma membrane. Progeny virions assemble in the cytoplasm at the surface of endoplasmic reticulum-derived membrane segments. Genome packaging initiates synchronously with the capsid assembly and completes through an aperture in the forming capsid. The genome-filled capsids acquire an outer membrane by budding into intracellular vesicles. EhV-201 infection induces a loss of surface protective layers from E. huxleyi cells, which enables the continuous release of virions by exocytosis.

A GP1BA Variant in a Czech Family with Monoallelic Bernard-Soulier Syndrome.

Bernard-Soulier syndrome (BSS) is a rare inherited disorder characterized by unusually large platelets, low platelet count, and prolonged bleeding time. BSS is usually inherited in an autosomal recessive (AR) mode of inheritance due to a deficiency of the GPIb-IX-V complex also known as the von Willebrand factor (VWF) receptor. We investigated a family with macrothrombocytopenia, a mild bleeding tendency, slightly lowered platelet aggregation tests, and suspected autosomal dominant (AD) inheritance. We have detected a heterozygous GP1BA likely pathogenic variant, causing monoallelic BSS. A germline GP1BA gene variant (NM_000173:c.98G > A:p.C33Y), segregating with the macrothrombocytopenia, was detected by whole-exome sequencing. In silico analysis of the protein structure of the novel GPIbα variant revealed a potential structural defect, which could impact proper protein folding and subsequent binding to VWF. Flow cytometry, immunoblot, and electron microscopy demonstrated further differences between p.C33Y GP1BA carriers and healthy controls. Here, we provide a detailed insight into its clinical presentation and phenotype. Moreover, the here described case first presents an mBSS patient with two previous ischemic strokes.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography.

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Targeting and modulating infarct macrophages with hemin formulated in designed lipid-based particles improves cardiac remodeling and function.

Uncontrolled activation of pro-inflammatory macrophages after myocardial infarction (MI) accelerates adverse left ventricular (LV) remodeling and dysfunction. Hemin, an iron-containing porphyrin, activates heme oxygenase-1 (HO-1), an enzyme with anti-inflammatory and cytoprotective properties. We sought to determine the effects of hemin formulated in a macrophage-targeted lipid-based carrier (denoted HA-LP) on LV remodeling and function after MI. Hemin encapsulation efficiency was ~100% at therapeutic dose levels. In vitro, hemin/HA-LP abolished TNF-α secretion from macrophages, whereas the same doses of free hemin and drug free HA-LP had no effect. Hemin/HA-LP polarized peritoneal and splenic macrophages toward M2 anti-inflammatory phenotype. We next induced MI in mice and allocated them to IV treatment with hemin/HA-LP (10mg/kg), drug free HA-LP, free hemin (10mg/kg) or saline, one day after MI. Active in vivo targeting to infarct macrophages was confirmed with HA-LP doped with PE-rhodamine. LV remodeling and function were assessed by echocardiography before, 7, and 30days after treatment. Significantly, hemin/HA-LP effectively and specifically targets infarct macrophages, switches infarct macrophages toward M2 anti-inflammatory phenotype, improves angiogenesis, reduces scar expansion and improves infarct-related regional function. In conclusion, macrophage-targeted lipid-based drug carriers with hemin switch macrophages into an anti-inflammatory phenotype, and improve infarct healing and repair. Our approach presents a novel strategy to modulate inflammation and improve infarct repair.

Loss of Macrophage Wnt Secretion Improves Remodeling and Function After Myocardial Infarction in Mice.

BACKGROUND: Macrophages and Wnt proteins (Wnts) are independently involved in cardiac development, response to cardiac injury, and repair. However, the role of macrophage-derived Wnts in the healing and repair of myocardial infarction (MI) is unknown. We sought to determine the role of macrophage Wnts in infarct repair. METHODS AND RESULTS: We show that the Wnt pathway is activated after MI in mice. Furthermore, we demonstrate that isolated infarct macrophages express distinct Wnt pathway components and are a source of noncanonical Wnts after MI. To determine the effect of macrophage Wnts on cardiac repair, we evaluated mice lacking the essential Wnt transporter Wntless (Wls) in myeloid cells. Significantly, Wntless-deficient macrophages presented a unique subset of M2-like macrophages with anti-inflammatory, reparative, and angiogenic properties. Serial echocardiography studies revealed that mice lacking macrophage Wnt secretion showed improved function and less remodeling 30 days after MI. Finally, mice lacking macrophage-Wntless had increased vascularization near the infarct site compared with controls. CONCLUSIONS: Macrophage-derived Wnts are implicated in adverse cardiac remodeling and dysfunction after MI. Together, macrophage Wnts could be a new therapeutic target to improve infarct healing and repair.

Injectable collagen implant improves survival, cardiac remodeling, and function in the early period after myocarditis in rats.

AIM: Despite clear evidence of immune system involvement in the pathogenesis of myocarditis, the treatment of myocarditis remains nonspecific and supportive. We sought to test the hypothesis that injection of a collagen-based implant into the inflamed myocardium would stabilize the left ventricular (LV) wall and prevent adverse remodeling and dysfunction. METHODS AND RESULTS: Autoimmune myocarditis was induced in 42 male Lewis rats. Development of myocarditis was evaluated and confirmed by serial echocardiography and cardiac magnetic resonance scans, LV wall thickening, global and regional LV wall motion abnormalities, and in some cases pericardial effusion. Sick animals were randomized to either injectable collagen implantation or saline injection into the anterior inflamed myocardium 14 days after immunization. Significantly, injectable collagen implantation improved 31-day survival compared with controls (85.7% vs 50%; P = .03). Furthermore, although injectable collagen significantly attenuated LV systolic and diastolic dilatation and preserved LV geometry and function, control animals developed significant LV dilatation and dysfunction. These favorable effects on LV remodeling were confirmed by postmortem morphometry. Significantly, the injectable collagen implant attenuated cardiomyocyte hypertrophy and infiltration of macrophages and lymphocytes into the myocardium. CONCLUSIONS: The present study shows, for the first time, that injectable collagen biomaterial improves survival and attenuates cardiac inflammation, cardiomyocyte hypertrophy, LV remodeling, and dysfunction in the early period after myocarditis in rats. Our findings suggest a new biomaterial-based strategy to ameliorate the devastating effects of myocarditis.

The origin of human mesenchymal stromal cells dictates their reparative properties.

BACKGROUND: Human mesenchymal stromal cells (hMSCs) from adipose cardiac tissue have attracted considerable interest in regard to cell-based therapies. We aimed to test the hypothesis that hMSCs from the heart and epicardial fat would be better cells for infarct repair. METHODS AND RESULTS: We isolated and grew hMSCs from patients with ischemic heart disease from 4 locations: epicardial fat, pericardial fat, subcutaneous fat, and the right atrium. Significantly, hMSCs from the right atrium and epicardial fat secreted the highest amounts of trophic and inflammatory cytokines, while hMSCs from pericardial and subcutaneous fat secreted the lowest. Relative expression of inflammation- and fibrosis-related genes was considerably higher in hMSCs from the right atrium and epicardial fat than in subcutaneous fat hMSCs. To determine the functional effects of hMSCs, we allocated rats to hMSC transplantation 7 days after myocardial infarction. Atrial hMSCs induced greatest infarct vascularization as well as highest inflammation score 27 days after transplantation. Surprisingly, cardiac dysfunction was worst after transplantation of hMSCs from atrium and epicardial fat and minimal after transplantation of hMSCs from subcutaneous fat. These findings were confirmed by using hMSC transplantation in immunocompromised mice after myocardial infarction. Notably, there was a correlation between tumor necrosis factor-α secretion from hMSCs and posttransplantation left ventricular remodeling and dysfunction. CONCLUSIONS: Because of their proinflammatory properties, hMSCs from the right atrium and epicardial fat of cardiac patients could impair heart function after myocardial infarction. Our findings might be relevant to autologous mesenchymal stromal cell therapy and development and progression of ischemic heart disease.

Macrophage subpopulations are essential for infarct repair with and without stem cell therapy.

OBJECTIVES: This study sought to investigate the hypothesis that the favorable effects of mesenchymal stromal cells (MSCs) on infarct repair are mediated by macrophages. BACKGROUND: The favorable effects of MSC therapy in myocardial infarction (MI) are complex and not fully understood. METHODS: We induced MI in mice and allocated them to bone marrow MSCs, mononuclear cells, or saline injection into the infarct, with and without early (4 h before MI) and late (3 days after MI) macrophage depletion. We then analyzed macrophage phenotype in the infarcted heart by flow cytometry and macrophage secretome in vitro. Left ventricular remodeling and global and regional function were assessed by echocardiography and speckle-tracking based strain imaging. RESULTS: The MSC therapy significantly increased the percentage of reparative M2 macrophages (F4/80(+)CD206(+)) in the infarcted myocardium, compared with mononuclear- and saline-treated hearts, 3 and 4 days after MI. Macrophage cytokine secretion, relevant to infarct healing and repair, was significantly increased after MSC therapy, or incubation with MSCs or MSC supernatant. Significantly, with and without MSC therapy, transient macrophage depletion increased mortality 30 days after MI. Furthermore, early macrophage depletion produced the greatest negative effect on infarct size and left ventricular remodeling and function, as well as a significant incidence of left ventricular thrombus formation. These deleterious effects were attenuated with macrophage restoration and MSC therapy. CONCLUSIONS: Some of the protective effects of MSCs on infarct repair are mediated by macrophages, which are essential for early healing and repair. Thus, targeting macrophages could be a novel strategy to improve infarct healing and repair.

Patient characteristics and cell source determine the number of isolated human cardiac progenitor cells.

BACKGROUND: The identification and isolation of human cardiac progenitor cells (hCPCs) offer new approaches for myocardial regeneration and repair. Still, the optimal source of human cardiac progenitor cells and the influence of patient characteristics on their number remain unclear. Using a novel method to isolate human cardiac progenitor cells, we aimed to define the optimal source and association between their number and patient characteristics. METHODS AND RESULTS: We developed a novel isolation method that produced viable cells (7 x 10(6)+/-6.53 x 10(5)/g) from various tissue samples obtained during heart surgery or endomyocardial biopsies (113 samples from 94 patients 23 to 80 years of age). The isolated cardiac cells were grown in culture with a stem cell expansion medium. According to fluorescence-activated cell sorting analysis, cultured cells derived from the right atrium generated higher amounts of c-kit(+) (24+/-2.5%) and Islet-1(+) cells (7%) in culture (mean of passages 1, 2, and 3) than did cultured cells from the left atrium (7.3+/-3.5%), right ventricle (4.1+/-1.6%), and left ventricle (9.7+/-3%; P=0.001). According to multivariable analysis, the right atrium as the cell source and female sex were associated with a higher number of c-kit(+) cells. There was no overlap between c-kit(+) and Islet-1 expression. In vitro assays of differentiation into osteoblasts, adipocytes, and myogenic lineage showed that the isolated human cardiac progenitor cells were multipotent. Finally, the cells were transplanted into infarcted myocardium of rats and generated myocardial grafts. CONCLUSIONS: Our results show that the right atrium is the best source for c-kit(+) and Islet-1 progenitors, with higher percentages of c-kit(+) cells being produced by women.

Prevascularization of cardiac patch on the omentum improves its therapeutic outcome.

The recent progress made in the bioengineering of cardiac patches offers a new therapeutic modality for regenerating the myocardium after myocardial infarction (MI). We present here a strategy for the engineering of a cardiac patch with mature vasculature by heterotopic transplantation onto the omentum. The patch was constructed by seeding neonatal cardiac cells with a mixture of prosurvival and angiogenic factors into an alginate scaffold capable of factor binding and sustained release. After 48 h in culture, the patch was vascularized for 7 days on the omentum, then explanted and transplanted onto infarcted rat hearts, 7 days after MI induction. When evaluated 28 days later, the vascularized cardiac patch showed structural and electrical integration into host myocardium. Moreover, the vascularized patch induced thicker scars, prevented further dilatation of the chamber and ventricular dysfunction. Thus, our study provides evidence that grafting prevascularized cardiac patch into infarct can improve cardiac function after MI.

Electromagnetic field at 15.95-16 Hz is cardio protective following acute myocardial infarction.

Previous studies have shown that pre-exposure of the heart to weak magnetic field reduces infarct size shortly after induction of myocardial ischemia. To investigate the role of AC magnetic field with a frequency of 15.95-16 Hz and 80 mT on left ventricular (LV) remodeling following chronic coronary occlusion and a short episode of ischemia followed by reperfusion (I/R). LV dimension and function were measured using echocardiography. Femur bone marrow was isolated and cells were phenotyped for endothelial linage and immuno stained for endothelial cells. The area at risk was measured using triphenyltetrazolium chloride staining. A significant reduction of 27% in shortening fraction (SF) was measured following acute myocardial infarction (AMI) compared with a 7% decrease in animals exposed to magnetic field (p < 0.04). A significantly higher number of colony forming units and endothelial progenitor cells were counted within the treated groups subjected to magnetic field (p < 0.02). Exposing the heart to magnetic field prior to reperfusion did not show any preservation either on SF or on infarct size. Magnetic field was protective in the AMI but not in the I/R model. The mechanisms underlying cardiac protection induced by AC magnetic field following chronic injury deserves further investigation.

Evaluation of a peritoneal-generated cardiac patch in a rat model of heterotopic heart transplantation.

Tissue engineering holds the promise of providing new solutions for heart transplant shortages and pediatric heart transplantation. The aim of this study was to evaluate the ability of a peritoneal-generated, tissue-engineered cardiac patch to replace damaged myocardium in a heterotopic heart transplant model. Fetal cardiac cells (1 x 10(6)/scaffold) from syngeneic Lewis rats were seeded into highly porous alginate scaffolds. The cell constructs were cultured in vitro for 4 days and then they were implanted into the rat peritoneal cavity for 1 week. During this time the peritoneal-implanted patches were vascularized and populated with myofibroblasts. They were harvested and their performance in an infrarenal heterotopic abdominal heart transplantation model was examined (n = 15). After transplantation and before reperfusion of the donor heart, a 5-mm left (n = 6) or right (n = 9) ventriculotomy was performed and the patch was sutured onto the donor heart to repair the defect. Echocardiographical studies carried out 1-2 weeks after transplantation showed normal LV function in seven of the eight hearts studied. After 1 month, visual examination of the grafted patch revealed no aneurysmal dilatation. Microscopic examination revealed, in most of the cardiac patches, a complete disappearance of the scaffold and its replacement by a consistent tissue composed of myofibroblasts embedded in collagen bundles. The cardiac patch was enriched with a relatively large number of infiltrating blood vessels. In conclusion, cardiac patches generated in the peritoneum were developed into consistent tissue patches with properties to seal and correct myocardial defects. Our study also offers a viable rat model for screening and evaluating new concepts in cardiac reconstruction and engineering.

The effects of peptide-based modification of alginate on left ventricular remodeling and function after myocardial infarction.

Adverse cardiac remodeling and dysfunction after myocardial infarction (MI) is associated with (BioLineRx, BL-1040 myocardial implant) excessive damage to the extracellular matrix. Biomaterials, such as the in situ-forming alginate hydrogel, provide temporary support and attenuate these processes. Here, we tested the effects of decorating alginate biomaterial with cell adhesion peptides, containing the sequences RGD and YIGSR, or a non-specific peptide (RGE), in terms of therapeutic outcome soon after MI. The biomaterial (i.e., both unmodified and peptide-modified alginate) solutions retained the ability to flow after cross-linking with calcium ions, and could be injected into 7-day infarcts, where they underwent phase transition into hydrogels. Serial echocardiography studies performed before and 60 days after treatment showed that alginate modification with the peptides reduced the therapeutical effects of the hydrogel, as revealed by the extent of scar thickness, left ventricle dilatation and function. Histology and immunohistochemistry revealed no significant differences in blood vessel density, scar thickness, myofibroblast or macrophage infiltration or cell proliferation between the experimental groups BioLineRx BL-1040 myocardial implant. Our studies thus reveal that the chemical and physical traits of the biomaterial can affect its therapeutical efficacy in attenuating left ventricle remodeling and function, post-MI.

Transplantation of genetically engineered cardiac fibroblasts producing recombinant human erythropoietin to repair the infarcted myocardium.

BACKGROUND: Erythropoietin possesses cellular protection properties. The aim of the present study was to test the hypothesis that in situ expression of recombinant human erythropoietin (rhEPO) would improve tissue repair in rat after myocardial infarction (MI). METHODS AND RESULTS: RhEPO-producing cardiac fibroblasts were generated ex vivo by transduction with retroviral vector. The anti-apoptotic effect of rhEPO-producing fibroblasts was evaluated by co-culture with rat neonatal cardiomyocytes exposed to H2O2-induced oxidative stress. Annexin V/PI assay and DAPI staining showed that compared with control, rhEPO forced expression markedly attenuated apoptosis and improved survival of cultured cardiomyocytes. To test the effect of rhEPO on the infarcted myocardium, Sprague-Dawley rats were subjected to permanent coronary artery occlusion, and rhEPO-producing fibroblasts, non-transduced fibroblasts, or saline, were injected into the scar tissue seven days after infarction. One month later, immunostaining identified rhEPO expression in the implanted engineered cells but not in controls. Compared with non-transduced fibroblasts or saline injection, implanted rhEPO-producing fibroblasts promoted vascularization in the scar, and prevented cell apoptosis. By two-dimensional echocardiography and postmortem morphometry, transplanted EPO-engineered fibroblasts did not prevent left ventricular (LV) dysfunction and adverse LV remodeling 5 and 9 weeks after MI. CONCLUSION: In situ expression of rhEPO enhances vascularization and reduces cell apoptosis in the infarcted myocardium. However, local EPO therapy is insufficient for functional improvement after MI in rat.

Effect of injectable alginate implant on cardiac remodeling and function after recent and old infarcts in rat.

BACKGROUND: Adverse cardiac remodeling and progression of heart failure after myocardial infarction are associated with excessive and continuous damage to the extracellular matrix. We hypothesized that injection of in situ-forming alginate hydrogel into recent and old infarcts would provide a temporary scaffold and attenuate adverse cardiac remodeling and dysfunction. METHODS AND RESULTS: We developed a novel absorbable biomaterial composed of calcium-crosslinked alginate solution, which displays low viscosity and, after injection into the infarct, undergoes phase transition into hydrogel. To determine the outcome of the biomaterial after injection, calcium-crosslinked biotin-labeled alginate was injected into the infarct 7 days after anterior myocardial infarction in rat. Serial histology studies showed in situ formation of alginate hydrogel implant, which occupied up to 50% of the scar area. The biomaterial was replaced by connective tissue within 6 weeks. Serial echocardiography studies before and 60 days after injection showed that injection of alginate biomaterial into recent (7 days) infarct increased scar thickness and attenuated left ventricular systolic and diastolic dilatation and dysfunction. These beneficial effects were comparable and sometimes superior to those achieved by neonatal cardiomyocyte transplantation. Moreover, injection of alginate biomaterial into old myocardial infarction (60 days) increased scar thickness and improved systolic and diastolic dysfunction. CONCLUSIONS: We show for the first time that injection of in situ-forming, bioabsorbable alginate hydrogel is an effective acellular strategy that prevents adverse cardiac remodeling and dysfunction in recent and old myocardial infarctions in rat.

Iron-oxide labeling and outcome of transplanted mesenchymal stem cells in the infarcted myocardium.

BACKGROUND: Cell labeling with superparamagnetic iron oxide (SPIO) nanoparticles enables noninvasive MRI and tracking of transplanted stem cells. We sought to determine whether mesenchymal stem cell (MSC) outcome is affected by SPIO labeling in a rat model of myocardial infarction. METHODS AND RESULTS: Rat MSCs were labeled with SPIO (ferumoxides; Endorem; Guerbet, Villepinte, France). By trypan-blue exclusion assay, almost 100% of the cells remained viable after labeling. Seven days after MI, rats were randomized to injections of 2x10(6) SPIO-labeled MSCs, 2x10(6) unlabeled MSCs, or saline. Labeled cells were visualized in the infarcted myocardium as large black spots by serial MRI studies throughout the 4-week follow-up. The presence of labeled cells was confirmed by iron staining and real-time polymerase chain reaction on postmortem specimens. At 4 weeks after transplantation, the site of cell injection was infiltrated by inflammatory cells. Costaining for iron and ED1 (resident macrophage marker) showed that the iron-positive cells were cardiac macrophages. By real-time polymerase chain reaction, the Y-chromosome-specific SRY DNA of MSCs from male donors was not detected in infarcted hearts of female recipients. Serial echocardiography studies at baseline and 4 weeks after cell transplantation showed that both unlabeled and labeled MSCs attenuated progressive left ventricular dilatation and dysfunction compared with controls. CONCLUSIONS: At 4 weeks after transplantation of SPIO-labeled MSCs, the transplanted cells are not present in the scar and the enhanced MRI signals arise from cardiac macrophages that engulfed the SPIO nanoparticles. However, both labeled and unlabeled cells attenuate left ventricular dilatation and dysfunction after myocardial infarction.

Human embryonic stem cell transplantation to repair the infarcted myocardium.

OBJECTIVE: To test the hypothesis that human embryonic stem cells (hESCs) can be guided to form new myocardium by transplantation into the normal or infarcted heart, and to assess the influence of hESC-derived cardiomyocytes (hESCMs) on cardiac function in a rat model of myocardial infarction (MI). METHODS: Undifferentiated hESCs (0.5-1x10(6)), human embryoid bodies (hEBs) (4-8 days; 0.5-1x10(6)), 0.1 mm pieces of embryonic stem-derived beating myocardial tissue, and phosphate-buffered saline (control) were injected into the normal or infarcted myocardium of athymic nude rats (n = 58) by direct injection into the muscle or into preimplanted three-dimensional alginate scaffold. By 2-4 weeks after transplantation, heart sections were examined to detect the human cells and differentiation with fluorescent in situ hybridisation, using DNA probes specific for human sex chromosomes and HLA-DR or HLA-ABC immunostaining. RESULTS: Microscopic examination showed transplanted human cells in the normal, and to a lesser extent in the infarcted myocardium (7/7 vs 2/6; p<0.05). The transplanted hESCs and hEBs rarely created new vessels and did not form new myocardium. Transplantation of hESCM tissue into normal heart produced islands of disorganised myofibres, fibrosis and, in a single case, a teratoma. However, transplantation of hESCMs into the infarcted myocardium did prevent post-MI dysfunction and scar thinning. CONCLUSIONS: Undifferentiated hESCs and hEBs are not directed to form new myocardium after transplantation into normal or infarcted heart and may create teratoma. Nevertheless, this study shows that hESC-derived cardiomyocyte transplantation can attenuate post-MI scar thinning and left ventricular dysfunction.

Ex vivo activated human macrophages improve healing, remodeling, and function of the infarcted heart.

BACKGROUND: Activated macrophages have a significant role in wound healing and damaged tissue repair. We sought to explore the ability of ex vivo activated macrophages to promote healing and repair of the infarcted myocardium. METHODS AND RESULTS: Human activated macrophage suspension (AMS) was prepared from a whole blood unit obtained from young donors in a closed sterile system and was activated by a novel method of hypo-osmotic shock. The AMS (approximately 4 x 10(5) cells) included up to 43% CD14-positive cells and was injected into the ischemic myocardium of rats (n=8) immediately after coronary artery ligation. The control group (n=9) was treated with saline injection. The human cells existed in the infarcted heart 4 to 7 days after injection, as indicated by histology, human growth hormone-specific polymerase chain reaction, and magnetic resonance imaging (MRI) tracking of iron oxide-nanoparticle-labeled cells. After 5 weeks, scar vessel density (+/-SE) (25+/-4 versus 10+/-1 per mm2; P<0.05), myofibroblast accumulation, and recruitment of resident monocytes and macrophages were greater in AMS-treated hearts compared with controls. Serial echocardiography studies, before and 5 weeks after injection, showed that AMS improved scar thickening (0.15+/-0.01 versus 0.11+/-0.01 cm; P<0.05), reduced left ventricular (LV) diastolic dilatation (0.87+/-0.02 versus 0.99+/-0.04 cm; P<0.05), and improved LV fractional shortening (31+/-2 versus 20+/-4%; P<0.05), compared with controls. CONCLUSIONS: Early after myocardial infarction, injection of AMS accelerates vascularization, tissue repair, and improves cardiac remodeling and function. Our work suggests a novel clinically relevant option to promote the repair of ischemic tissue.

Evaluation of the pro-angiogenic effect of factor XIII in heterotopic mouse heart allografts and FXIII-deficient mice.

Thrombin-activated Factor XIII (FXIIIa), a plasma transglutaminase, stabilizes fibrin clots by crosslinking fibrin chains. FXIIIa was previously shown by us to exhibit proangiogenic activity associated with downregulation of thrombospondin-1, phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2), and upregulation of c-Jun. In the current study, we evaluated the proangiogenic effect of FXIIIa in two murine models: a neonatal heterotopic cardiac allograft model in normal mice, and a Matrigel plug model in FXIII-deficient mice. In the neonatal cardiac allograft model, the number of new vessels as well as graft viability (contractile performance) was significantly higher in FXIIIa-injected animals than in controls. A significant increase in the level of c-Jun mRNA and a significant decrease in the level of TSP-1 mRNA were observed in heart allografts treated with FXIIIa. A marked decrease in TSP-1 protein expression was observed within the endothelial cells of hearts treated with FXIIIa. In the Matrigel plug model, FXIII-deficient mice showed a significantly decreased number of new vessels compared to that of the control mice, and the number of vessels almost reached normal levels following addition of FXIIIa. The results of this study provide substantial in vivo evidence for the proangiogenic activity of FXIIIa.

Low-intensity ultrasound induces angiogenesis in rat hind-limb ischemia.

We investigated the effect of low-intensity ultrasound (US) on tissue blood flow and angiogenesis after limb ischemia in vivo. Rats underwent surgical ligation of the femoral or the iliac arteries. Half the animals were exposed to low-intensity US (0.05 W/cm2) during three consecutive sessions. At 3 weeks postsurgery, limb perfusion was assessed using laser Doppler and angiography. Immunostaining and vascular endothelial growth factor (VEGF) messenger ribonucleic acid (mRNA) expression were performed 7 d postsurgery. US irradiation significantly improved limb perfusion in both ischemic models (p = 0.04). Angiography showed increased blood vessels in the moderate ischemia (p = 0.01), but not in the severe ischemia (p = 0.19). Histology demonstrated a significantly higher number of blood vessels and proliferating cells in US-irradiated moderate and severe ischemia (p = 0.002 and p = 0.03, respectively). VEGF mRNA was significantly higher in moderate ischemia (p = 0.02). No differences in apoptotic cell death were evident in the models. Low-intensity US significantly improved tissue blood flow and angiogenesis, irrespective of the extent of the ischemia. (E-mail: ).