Fli1+ cells transcriptional analysis reveals an Lmo2-Prdm16 axis in angiogenesis.

A network of molecular factors drives the development, differentiation, and maintenance of endothelial cells. Friend leukemia integration 1 transcription factor (FLI1) is a bona fide marker of endothelial cells during early development. In zebrafish Tg(fli1:EGFP)y1 , we identified two endothelial cell populations, high-fli1+ and low-fli1+, by the intensity of green fluorescent protein signal. By comparing RNA-sequencing analysis of non-fli1 expressing cells (fli1-) with these two (fli1+) cell populations, we identified several up-regulated genes, not previously recognized as important, during endothelial development. Compared with fli1- and low-fli1+ cells, high-fli1+ cells showed up-regulated expression of the zinc finger transcription factor PRDI-BF1 and RIZ homology domain containing 16 (prdm16). Prdm16 knockdown (KD) by morpholino in the zebrafish larva was associated with impaired angiogenesis and increased number of low-fli1+ cells at the expense of high-fli1+ cells. In addition, PRDM16 KD in endothelial cells derived from human-induced pluripotent stem cells impaired their differentiation and migration in vitro. Moreover, zebrafish mutants (mut) with loss of function for the oncogene LIM domain only 2 (lmo2) also showed reduced prdm16 gene expression combined with impaired angiogenesis. Prdm16 expression was reduced further in endothelial (CD31+) cells compared with CD31- cells isolated from lmo2-mutants (lmo2-mut) embryos. Chromatin immunoprecipitation-PCR demonstrated that Lmo2 binds to the promoter and directly regulates the transcription of prdm16 This work unveils a mechanism by which prdm16 expression is activated in endothelial cells by Lmo2 and highlights a possible therapeutic pathway by which to modulate endothelial cell growth and repair.

Telomerase therapy reverses vascular senescence and extends lifespan in progeria mice.

AIMS: Hutchinson-Gilford progeria syndrome (HGPS) is an accelerated ageing syndrome associated with premature vascular disease and death due to heart attack and stroke. In HGPS a mutation in lamin A (progerin) alters nuclear morphology and gene expression. Current therapy increases the lifespan of these children only modestly. Thus, greater understanding of the underlying mechanisms of HGPS is required to improve therapy. Endothelial cells (ECs) differentiated from induced pluripotent stem cells (iPSCs) derived from these patients exhibit hallmarks of senescence including replication arrest, increased expression of inflammatory markers, DNA damage, and telomere erosion. We hypothesized that correction of shortened telomeres may reverse these measures of vascular ageing. METHODS AND RESULTS: We generated ECs from iPSCs belonging to children with HGPS and their unaffected parents. Telomerase mRNA (hTERT) was used to treat HGPS ECs. Endothelial morphology and functions were assessed, as well as proteomic and transcriptional profiles with attention to inflammatory markers, DNA damage, and EC identity genes. In a mouse model of HGPS, we assessed the effects of lentiviral transfection of mTERT on measures of senescence, focusing on the EC phenotype in various organs. hTERT treatment of human HGPS ECs improved replicative capacity; restored endothelial functions such as nitric oxide generation, acetylated low-density lipoprotein uptake and angiogenesis; and reduced the elaboration of inflammatory cytokines. In addition, hTERT treatment improved cellular and nuclear morphology, in association with a normalization of the transcriptional profile, effects that may be mediated in part by a reduction in progerin expression and an increase in sirtuin 1 (SIRT1). Progeria mice treated with mTERT lentivirus manifested similar improvements, with a reduction in inflammatory and DNA damage markers and increased SIRT1 in their vasculature and other organs. Furthermore, mTERT therapy increased the lifespan of HGPS mice. CONCLUSION: Vascular rejuvenation using telomerase mRNA is a promising approach for progeria and other age-related diseases.

TBX20 Regulates Angiogenesis Through the Prokineticin 2-Prokineticin Receptor 1 Pathway.

BACKGROUND: Angiogenesis is integral for embryogenesis, and targeting angiogenesis improves the outcome of many pathological conditions in patients. TBX20 is a crucial transcription factor for embryonic development, and its deficiency is associated with congenital heart disease. However, the role of TBX20 in angiogenesis has not been described. METHODS: Loss- and gain-of-function approaches were used to explore the role of TBX20 in angiogenesis both in vitro and in vivo. Angiogenesis gene array was used to identify key downstream targets of TBX20. RESULTS: Unbiased gene array survey showed that TBX20 knockdown profoundly reduced angiogenesis-associated PROK2 (prokineticin 2) gene expression. Indeed, loss of TBX20 hindered endothelial cell migration and in vitro angiogenesis. In a murine angiogenesis model using subcutaneously implanted Matrigel plugs, we observed that TBX20 deficiency markedly reduced PROK2 expression and restricted intraplug angiogenesis. Furthermore, recombinant PROK2 administration enhanced angiogenesis and blood flow recovery in murine hind-limb ischemia. In zebrafish, transient knockdown of tbx20 by morpholino antisense oligos or genetic disruption of tbx20 by CRISPR/Cas9 impaired angiogenesis. Furthermore, loss of prok2 or its cognate receptor prokr1a also limited angiogenesis. In contrast, overexpression of prok2 or prokr1a rescued the impaired angiogenesis in tbx20-deficient animals. CONCLUSIONS: Our study identifies TBX20 as a novel transcription factor regulating angiogenesis through the PROK2-PROKR1 (prokineticin receptor 1) pathway in both development and disease and reveals a novel mode of angiogenic regulation whereby the TBX20-PROK2-PROKR1 signaling cascade may act as a "biological capacitor" to relay and sustain the proangiogenic effect of vascular endothelial growth factor. This pathway may be a therapeutic target in the treatment of diseases with dysregulated angiogenesis.

Lmo2 (LIM-Domain-Only 2) Modulates Sphk1 (Sphingosine Kinase) and Promotes Endothelial Cell Migration.

OBJECTIVE: Lmo (LIM-domain-only)2 transcription factor is involved in hematopoiesis and vascular remodeling. Sphk (sphingosine kinase)1 phosphorylates sphingosine to S1P (sphingosine-1-phosphate). We hypothesized that Lmo2 regulates Sphk1 to promote endothelial cell (EC) migration and vascular development. APPROACH AND RESULTS: Lmo2 and Sphk1 knockdown (KD) were performed in Tg(fli1:EGFP) y1 zebrafish and in human umbilical vein EC. Rescue of phenotypes or overexpression of these factors were achieved using mRNA encoding Lmo2 or Sphk1. EC proliferation in vivo was assessed by BrdU (bromodeoxyuridine) immunostaining and fluorescence-activated cell sorter analysis of dissociated Tg(fli1:EGFP) y1 embryos. Cell migration was assessed by scratch assay in human umbilical vein EC and mouse aortic rings. Lmo2 interactions with Sphk1 promoter were assessed by ChIP-PCR (chromatin immunoprecipitation-polymerase chain reaction). Lmo2 or Sphk1 KD reduced number and length of intersegmental vessels. There was no reduction in the numbers of GFP+ (green fluorescent protein) ECs after Lmo2 KD. However, reduced numbers of BrdU+GFP+ nuclei were observed along the dysmorphic intersegmental vessels, accumulating instead at the sprouting origin of the intersegmental vessels. This anomaly was likely because of impaired EC migration, which was confirmed in migration assays using Lmo2 KD human umbilical vein ECs and mouse aortic rings. Both in vivo and in vitro, Lmo2 KD reduced Sphk1 gene expression, associated with less Lmo2 binding to the Sphk1 promoter as assessed by ChIP-PCR. Sphk1 mRNA rescued the Lmo2 KD phenotype. CONCLUSIONS: Our data showed that Lmo2 is necessary for Sphk1 gene expression in ECs. Lmo2 KD reduced Lmo2-Sphk1 gene interaction, impaired intersegmental vessels formation, and reduced cell migration. We identified for the first time Sphk1 as downstream effector of Lmo2.

Cardiomyocyte proliferation in zebrafish and mammals: lessons for human disease.

Cardiomyocytes proliferate profusely during early development and for a brief period after birth in mammals. Within a month after birth, this proliferative capability is dramatically reduced in mammals unlike lower vertebrates where it persists into adult life. The zebrafish, for example, retains the ability to regenerate the apex of the heart following resection by a mechanism predominantly driven by cardiomyocyte proliferation. Differences in proliferative capacity of cardiomyocytes in adulthood between mammals and lower vertebrates are closely liked to ontogenetic or phylogenetic factors. Elucidation of these factors has the potential to provide enormous benefits if they lead to the development of therapeutic strategies that facilitate cardiomyocyte proliferation. In this review, we highlight the differences between Mammalian and Zebrafish cardiomyocytes, which could explain at least in part the different proliferative capacities in these two species. We discuss the advantages of the zebrafish as a model of cardiomyocyte proliferation, particularly at the embryonic stage. We also identify a number of key molecular pathways with potential to reveal key steps in switching cardiomyocytes from a quiescent to a proliferative phenotype.

CDK9 and its repressor LARP7 modulate cardiomyocyte proliferation and response to injury in the zebrafish heart.

Cyclin dependent kinase (Cdk)9 acts through the positive transcription elongation factor-b (P-TEFb) complex to activate and expand transcription through RNA polymerase II. It has also been shown to regulate cardiomyocyte hypertrophy, with recent evidence linking it to cardiomyocyte proliferation. We hypothesised that modification of CDK9 activity could both impair and enhance the cardiac response to injury by modifying cardiomyocyte proliferation. Cdk9 expression and activity were inhibited in the zebrafish (Danio rerio) embryo. We show that dephosphorylation of residue Ser2 on the C-terminal domain of RNA polymerase II is associated with impaired cardiac structure and function, and cardiomyocyte proliferation and also results in impaired functional recovery following cardiac laser injury. In contrast, de-repression of Cdk9 activity, through knockdown of La-related protein (Larp7) increases phosphorylation of Ser2 in RNA polymerase II and increases cardiomyocyte proliferation. Larp7 knockdown rescued the structural and functional phenotype associated with knockdown of Cdk9. The balance of Cdk9 and Larp7 plays a key role in cardiomyocyte proliferation and response to injury. Larp7 represents a potentially novel therapeutic target to promote cardiomyocyte proliferation and recovery from injury.

Temporal cohesion of the structural, functional and molecular characteristics of the developing zebrafish heart.

Heart formation is a complex, dynamic and highly coordinated process of molecular, morphogenetic and functional factors with each interacting and contributing to formation of the mature organ. Cardiac abnormalities in early life can be lethal in mammals but not in the zebrafish embryo which has been widely used to study the developing heart. While early cardiac development in the zebrafish has been well characterized, functional changes during development and how these relate to architectural, cellular and molecular aspects of development have not been well described previously. To address this we have carefully characterised cardiac structure, function, cardiomyocyte proliferation and cardiac-specific gene expression between 48 and 120 hpf in the zebrafish. We show that the zebrafish heart increases in volume and changes shape significantly between 48 and 72 hpf accompanied by a 40% increase in cardiomyocyte number. Between 96 and 120 hpf, while external heart expansion slows, there is rapid formation of a mature and extensive trabecular network within the ventricle chamber. While ejection fraction does not change during the course of development other determinants of contractile function increase significantly particularly between 72 and 96 hpf leading to an increase in cardinal vein blood flow. This study has revealed a number of novel aspects of cardiac developmental dynamics with striking temporal orchestration of structure and function within the first few days of development. These changes are associated with changes in expression of developmental and maturational genes. This study provides important insights into the complex temporal relationship between structure and function of the developing zebrafish heart.

Macrophages trigger cardiomyocyte proliferation by increasing epicardial vegfaa expression during larval zebrafish heart regeneration.

Cardiac injury leads to the loss of cardiomyocytes, which are rapidly replaced by the proliferation of the surviving cells in zebrafish, but not in mammals. In both the regenerative zebrafish and non-regenerative mammals, cardiac injury induces a sustained macrophage response. Macrophages are required for cardiomyocyte proliferation during zebrafish cardiac regeneration, but the mechanisms whereby macrophages facilitate this crucial process are fundamentally unknown. Using heartbeat-synchronized live imaging, RNA sequencing, and macrophage-null genotypes in the larval zebrafish cardiac injury model, we characterize macrophage function and reveal that these cells activate the epicardium, inducing cardiomyocyte proliferation. Mechanistically, macrophages are specifically recruited to the epicardial-myocardial niche, triggering the expansion of the epicardium, which upregulates vegfaa expression to induce cardiomyocyte proliferation. Our data suggest that epicardial Vegfaa augments a developmental cardiac growth pathway via increased endocardial notch signaling. The identification of this macrophage-dependent mechanism of cardiac regeneration highlights immunomodulation as a potential strategy for enhancing mammalian cardiac repair.

Nuclear S-nitrosylation impacts tissue regeneration in zebrafish.

Despite the importance of nitric oxide signaling in multiple biological processes, its role in tissue regeneration remains largely unexplored. Here, we provide evidence that inducible nitric oxide synthase (iNos) translocates to the nucleus during zebrafish tailfin regeneration and is associated with alterations in the nuclear S-nitrosylated proteome. iNos inhibitors or nitric oxide scavengers reduce protein S-nitrosylation and impair tailfin regeneration. Liquid chromatography/tandem mass spectrometry reveals an increase of up to 11-fold in the number of S-nitrosylated proteins during regeneration. Among these, Kdm1a, a well-known epigenetic modifier, is S-nitrosylated on Cys334. This alters Kdm1a binding to the CoRest complex, thus impairing its H3K4 demethylase activity, which is a response specific to the endothelial compartment. Rescue experiments show S-nitrosylation is essential for tailfin regeneration, and we identify downstream endothelial targets of Kdm1a S-nitrosylation. In this work, we define S-nitrosylation as an essential post-translational modification in tissue regeneration.

Adiponectinemia controls pro-angiogenic cell therapy.

Angiogenic cell therapy with the transplantation of endothelial progenitor cells (EPC) or bone marrow mononuclear cells (BM-MNC) receives considerable attention as an approach to revascularize ischemic tissues. Adiponectin is a circulating hormone produced by the apM1 gene in adipocytes. Adiponectin modulates lipid metabolism and obesity, and it was recently found to promote physiological angiogenesis in response to ischemia. Patients with multiple cardiovascular disease risk factors or myocardial infarction may benefit from progenitor cell therapy, but they display depressed adiponectinemia. We hypothesized that adiponectin stimulation of transplanted cells is critical for their pro-angiogenic function. We aimed to establish whether adiponectinemia in the cell donor or in the cell recipient determines the success of pro-angiogenic cell therapy. In vitro, we found that conditioned media derived from wild-type adipocytes (adipo-CM) or purified adiponectin strongly enhanced BM-MNC survival and proliferation and stimulated EPC differentiation, whereas adipo-CM from apM1-/- adipocytes was one-half less effective. On the other hand, wild-type and apM1-/- BM-MNC displayed similar resistance to apoptosis and proliferation rates. In vivo, wild-type, and apM1-/- BM-MNC induced similar angiogenic reactions in wild-type ischemic hindlimbs. In contrast, wild-type BM-MNC had much diminished effects in apM1-/- ischemic hindlimbs. We concluded that adiponectin enhances BM-MNC survival and proliferation, and adiponectinemia in the cell therapy recipient is essential for the pro-angiogenic benefits of cell therapy. These observations imply that progenitor cell transplantation might only induce angiogenesis in patients with high adiponectinemia.

Coadministration of endothelial and smooth muscle progenitor cells enhances the efficiency of proangiogenic cell-based therapy.

Cell-based therapy is a promising approach designed to enhance neovascularization and function of ischemic tissues. Interaction between endothelial and smooth muscle cells regulates vessels development and remodeling and is required for the formation of a mature and functional vascular network. Therefore, we assessed whether coadministration of endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs) can increase the efficiency of cell therapy. Unilateral hindlimb ischemia was surgically induced in athymic nude mice treated with or without intravenous injection of EPCs (0.5 x 10(6)), SMPCs (0.5 x 10(6)) and EPCs+SMPCs (0.25 x 10(6)+0.25 x 10(6)). Vessel density and foot perfusion were increased in mice treated with EPCs+SMPCs compared to animals receiving EPCs alone or SMPCs alone (P<0.001). In addition, capillary and arteriolar densities were enhanced in EPC+SMPC-treated mice compared to SMPC and EPC groups (P<0.01). We next examined the role of Ang-1/Tie2 signaling in the beneficial effect of EPC and SMPC coadministration. Small interfering RNA directed against Ang-1-producing SMPCs or Tie2-expressing EPCs blocked vascular network formation in Matrigel coculture assays, reduced the rate of incorporated EPCs within vascular structure, and abrogated the efficiency of cell therapy. Production of Ang-1 by SMPCs activates Tie2-expressing EPCs, resulting in increase of EPC survival and formation of a stable vascular network. Subsequently, the efficiency of EPC- and SMPC-based cotherapy is markedly increased. Therefore, coadministration of different types of vascular progenitor cells may constitute a novel therapeutic strategy for improving the treatment of ischemic diseases.

Exercise promotes angiogenesis and improves beta-adrenergic receptor signalling in the post-ischaemic failing rat heart.

AIMS: We investigated whether exercise training could promote angiogenesis and improve blood perfusion and left ventricular (LV) remodelling of the post-myocardial infarction (MI) failing heart. We also explored the contribution of ameliorated beta-adrenergic receptor signalling and function on the overall improvement of cardiac contractility reserve induced by exercise. METHODS AND RESULTS: Adult Wistar male rats were randomly assigned to one of four experimental groups. Sham-operated and post-MI heart failure (HF) rats were housed under sedentary conditions or assigned to 10-weeks of a treadmill exercise protocol. At 4 weeks after MI, sedentary HF rats showed LV eccentric hypertrophy, marked increase of LV diameters associated with severely impaired fractional shortening (14 +/- 5%), increased LV end diastolic pressure (20.9 +/- 2.6 mmHg), and pulmonary congestion. In addition, cardiac contractile responses to adrenergic stimulation were significantly blunted. In trained HF rats, exercise was able to (i) reactivate the cardiac vascular endothelial growth factor pathway with a concurrent enhancement of myocardial angiogenesis, (ii) significantly increase myocardial perfusion and coronary reserve, (iii) reduce cardiac diameters, and (iv) improve LV contractility in response to adrenergic stimulation. This latter finding was also associated with a significant improvement of cardiac beta-adrenergic receptor downregulation and desensitization. CONCLUSIONS: Our data indicate that exercise favourably affects angiogenesis and improves LV remodelling and contractility reserve in a rat model of severe chronic HF.

Dysfunction of iPSC-derived endothelial cells in human Hutchinson-Gilford progeria syndrome.

Children with Hutchinson-Gilford progeria syndrome (HGPS) succumb to myocardial infarction and stroke in their teen years. Endothelial dysfunction is an early event in more common forms of atherosclerosis. Endothelial pathobiology may contribute to HGPS, but a comprehensive characterization of endothelial function in HGPS has not been performed. iPSCs derived from fibroblasts of HGPS patients or unaffected relatives were differentiated into endothelial cells (ECs). Immunofluorescent signal of the pluripotent stem cell markers SSEA4, Oct4, Sox2 and TRAI-60 was similar in HGPS or control iPSCs. Following the differentiation, FACS analysis and immunocytochemistry for CD31 and CD144 revealed a smaller percentage of ECs from HGPS iPSCs. Immunostaining for Lamin A revealed nuclear dysmorphology in HGPS iPSC-ECs. Furthermore, these cells were significantly larger and rounded, and they proliferated less, features which are typical of senescent endothelial cells. HGPS iPSC-ECs manifested less Dil-Ac-LDL uptake; less DAF-2DA staining for nitric oxide generation and formed fewer networks in matrigel in vitro. In immunodeficient mice injected with iPSC-ECs, HGPS iPSC-ECs generated a sparser vascular network compared to the control, with reduced capillary number. Telomere length (T/S ratio) of HGPS iPSC-EC was reduced as assessed by mmqPCR. iPSC-ECs derived from HGPS patients have dysmorphic appearance, abnormal nuclear morphology, shortened telomeres, reduced replicative capacity and impaired functions in vitro and in vivo. Targeting the endothelial abnormality in patients with HGPS may provide a new therapeutic avenue for the treatment of this condition. Abbreviations: HGPS: Hutchinson-Gilford progeria syndrome; ZMPSTE24: Zinc metallopeptidase STE24; FTI: Farnesyltransferase inhibitors; VSMCs: Vascular smooth muscle cells; iPSC: Induced pluripotent stem cells; EC: Endothelial cells; hTERT: Human telomerase reverse transcriptase; VEGF: vascular endothelial growth factor; DAF-FM DA: 3-Amino, 4-aminomethyl-2',7'-difluorofluorescein diacetate; BMP4: Bone Morphogenetic Protein 4; mmqPCR: mono chrome multiplex PCR; SCG: single-copy gene; CSI: Cell shape index.

Discovery of novel determinants of endothelial lineage using chimeric heterokaryons.

We wish to identify determinants of endothelial lineage. Murine embryonic stem cells (mESC) were fused with human endothelial cells in stable, non-dividing, heterokaryons. Using RNA-seq, it is possible to discriminate between human and mouse transcripts in these chimeric heterokaryons. We observed a temporal pattern of gene expression in the ESCs of the heterokaryons that recapitulated ontogeny, with early mesodermal factors being expressed before mature endothelial genes. A set of transcriptional factors not known to be involved in endothelial development was upregulated, one of which was POU class 3 homeobox 2 (Pou3f2). We confirmed its importance in differentiation to endothelial lineage via loss- and gain-of-function (LOF and GOF). Its role in vascular development was validated in zebrafish embryos using morpholino oligonucleotides. These studies provide a systematic and mechanistic approach for identifying key regulators in directed differentiation of pluripotent stem cells to somatic cell lineages.

Effects of Cyclin Dependent Kinase 9 inhibition on zebrafish larvae.

CDK9 is a known regulator of cellular transcription, growth and proliferation. Small molecule inhibitors are currently being developed and assessed in clinical trials as anti-cancer drugs. The zebrafish embryo provides an ideal model to explore the effects of CDK9 inhibition in-vivo. This has not been adequately explored previously at the level of a whole organism. We have compared and contrasted the effects of pharmacological and molecular inhibition of CDK9 on somatic growth, apoptosis and cellular proliferation in zebrafish larvae between 0 to 120 hours post fertilisation (hpf) using flavopiridol, a selective CDK9 antagonist, and CDK9-targeting morpholino. We demonstrate that the inhibition of CDK9 diminishes cellular proliferation and increases apoptosis. Subsequently, it affects somatic growth and development of a number of key embryonic structures including the brain, heart, eye and blood vessels. For the first time, we have localized CDK9 at a subcellular level in whole-mounted larvae. This works shows, at a high-throughput level, that CDK9 clearly plays a fundamental role in early cellular growth and proliferation.

LIM Domain Only 2 Regulates Endothelial Proliferation, Angiogenesis, and Tissue Regeneration.

BACKGROUND: LIM domain only 2 (LMO2, human gene) is a key transcription factor that regulates hematopoiesis and vascular development. However, its role in adult endothelial function has been incompletely characterized. METHODS AND RESULTS: In vitro loss- and gain-of-function studies on LMO2 were performed in human umbilical vein endothelial cells with lentiviral overexpression or short hairpin RNA knockdown (KD) of LMO2, respectively. LMO2 KD significantly impaired endothelial proliferation. LMO2 controls endothelial G1/S transition through transcriptional regulation of cyclin-dependent kinase 2 and 4 as determined by reverse transcription polymerase chain reaction (PCR), western blot, and chromatin immunoprecipitation, and also influences the expression of Cyclin D1 and Cyclin A1. LMO2 KD also impaired angiogenesis by reducing transforming growth factor-ß (TGF-ß) expression, whereas supplementation of exogenous TGF-ß restored defective network formation in LMO2 KD human umbilical vein endothelial cells. In a zebrafish model of caudal fin regeneration, RT-PCR revealed that the lmo2 (zebrafish gene) gene was upregulated at day 5 postresection. The KD of lmo2 by vivo-morpholino injections in adult Tg(fli1:egfp)y1 zebrafish reduced 5-bromo-2'-deoxyuridine incorporation in endothelial cells, impaired neoangiogenesis in the resected caudal fin, and substantially delayed fin regeneration. CONCLUSIONS: The transcriptional factor LMO2 regulates endothelial proliferation and angiogenesis in vitro. Furthermore, LMO2 is required for angiogenesis and tissue healing in vivo. Thus, LMO2 is a critical determinant of vascular and tissue regeneration.

Modulation of 3-hydroxy-3-methylglutaryl-CoA reductase gene expression by CuZn superoxide dismutase in human fibroblasts and HepG2 cells.

The homeostasis of intracellular cholesterol in animal cells is highly regulated by a complex system in which the microsomal rate-limiting enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase plays a key role in cholesterol synthesis. Substantial evidence has demonstrated that the cytosolic antioxidant enzyme CuZn superoxide dismutase (SOD1) inhibits the HMG-CoA reductase activity in rat hepatocytes and in human fibroblasts by decreasing cholesterol synthesis. Although these data suggest that SOD1 exerts a physiological role in cholesterol metabolism, it is still unclear whether the decrease of HMG-CoA reductase activity is mediated by transcriptional or by posttranscriptional events. The results of the present study, obtained by one-step RT-PCR assay, demonstrated that both SOD1 and the metal-free form of enzyme (Apo SOD1) inhibit HMG-CoA reductase gene expression in hepatocarcinoma HepG2 cells, in normal human fibroblasts, and in fibroblasts of subjects affected by familiar hypercholesterolemia. Accordingly, SOD1 could be used as a potential agent in the treatment of hypercholesterolemia, even in subjects lacking a functional LDL receptor pathway.

Targeted laser ablation of the zebrafish larval heart induces models of heart block, valvular regurgitation, and outflow tract obstruction.

Mammalian models of cardiac disease have provided unique and important insights into human disease but have become increasingly challenging to produce. The zebrafish could provide inexpensive high-throughput models of cardiac injury and repair. We used a highly targeted laser, synchronized to fire at specific phases of the cardiac cycle, to induce regional injury to the ventricle, atrioventricular (AV) cushion, and bulbus arteriosus (BA). We assessed the impact of laser injury on hearts of zebrafish early larvae at 72 h postfertilization, to different regions, recording the effects on ejection fraction (EF), heart rate (HR), and blood flow at 2 and 24 h postinjury (hpi). Laser injury to the apex, midzone, and outflow regions of the ventricle resulted in reductions of the ventricle EF at 2 hpi with full recovery of function by 24 hpi. Laser injury to the ventricle, close to the AV cushion, was more likely to cause bradycardia and atrial-ventricular dysfunction, suggestive of an electrical conduction block. At 2 hpi, direct injury to the AV cushion resulted in marked regurgitation of blood from the ventricle to the atrium. Laser injury to the BA caused temporary outflow tract obstruction with cessation of ventricle contraction and circulation. Despite such damage, 80% of embryos showed complete recovery of the HR and function within 24 h of laser injury. Precision laser injury to key structures in the zebrafish developing heart provides a range of potentially useful models of hemodynamic overload, injury, and repair.

Laser-targeted ablation of the zebrafish embryonic ventricle: a novel model of cardiac injury and repair.

BACKGROUND: While the adult zebrafish (Danio rerio) heart demonstrates a remarkable capacity for self-renewal following apical resection little is known about the response to injury in the embryonic heart. METHODS: Injury to the beating zebrafish embryo heart was induced by laser using a transgenic zebrafish expressing cardiomyocyte specific green fluorescent protein. Changes in ejection fraction (EF), heart rate (HR), and caudal vein blood flow (CVBF) assessed by video capture techniques were assessed at 2, 24 and 48 h post-laser. Change in total and mitotic ventricular cardiomyocyte number following laser injury was also assessed by counting respectively DAPI (VCt) and Phospho-histone H3 (VCm) positive nuclei in isolated hearts using confocal microscopy. RESULTS: Laser injury to the ventricle resulted in bradycardia and mild bleeding into the pericardium. At 2 h post-laser injury, there was a significant reduction in cardiac performance in lasered-hearts compared with controls (HR 117 ± 11 vs 167 ± 9 bpm, p ≤ 0.001; EF 14.1 ± 1.8 vs 20.1 ± 1.3%, p ≤ 0.001; CVBF 103 ± 15 vs 316 ± 13 µms(-1), p ≤ 0.001, respectively). Isolated hearts showed a significant reduction in VCt at 2 h post-laser compared to controls (195 ± 15 vs 238 ± 15, p ≤ 0.05). Histology showed necrosis and apoptosis (TUNEL assay) at the site of laser injury. At 24 h post-laser cardiac performance and VCt had recovered fully to control levels. Pretreatment with the cell-cycle inhibitor, aphidicolin, significantly inhibited functional recovery of the ventricle accompanied by a significant inhibition of cardiomyocyte proliferation. CONCLUSIONS: Laser-targeted injury of the zebrafish embryonic heart is a novel and reproducible model of cardiac injury and repair suitable for pharmacological and molecular studies.