scholarly journals A simple adeno-associated virus-based approach for the generation of cardiac genetic models in rats

F1000Research ◽  
2020 ◽  
Vol 9 ◽  
pp. 1441
Author(s):  
Michal Schlesinger-Laufer ◽  
Guy Douvdevany ◽  
Lilac Haimovich-Caspi ◽  
Yaniv Zohar ◽  
Rona Shofty ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Rat Heart ◽  
Intraperitoneal Injection ◽  
Disease Modeling ◽  
Genetic Manipulation ◽  
Troponin T ◽  
Adeno Associated Virus ◽  
Specific Level ◽  
Major Health Problem ◽  
Single Intraperitoneal Injection

Background: Heart failure is a major health problem and progress in this field relies on better understanding of the mechanisms and development of novel therapeutics using animal models. The rat may be preferable to the mouse as a cardiovascular disease model due to its closer physiology to humans and due to its large size that facilitates surgical and monitoring procedures. However, unlike the mouse, genetic manipulation of the rat genome is challenging. Methods: Here we developed a simple, refined, and robust cardiac-specific rat transgenic model based on an adeno-associated virus (AAV) 9 containing a cardiac troponin T promoter. This model uses a single intraperitoneal injection of AAV and does not require special expertise or equipment. Results: We characterize the AAV dose required to achieve a high cardiac specific level of expression of a transgene in the rat heart using a single intraperitoneal injection to neonates. We show that at this AAV dose GFP expression does not result in hypertrophy, a change in cardiac function or other evidence for toxicity. Conclusions: The model shown here allows easy and fast transgenic based disease modeling of cardiovascular disease in the rat heart, and can also potentially be expanded to deliver Cas9 and gRNAs or to deliver small hairpin (sh)RNAs to also achieve gene knockouts and knockdown in the rat heart.

scholarly journals Rapid target validation in a Cas9-inducible hiPSC derived kidney model

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yasaman Shamshirgaran ◽  
Anna Jonebring ◽  
Anna Svensson ◽  
Isabelle Leefa ◽  
Mohammad Bohlooly-Y ◽  
...  
Keyword(s):  
High Efficiency ◽  
Disease Modeling ◽  
Genetic Manipulation ◽  
Disease Model ◽  
Genetically Engineered ◽  
Model Systems ◽  
Target Validation ◽  
Direct Differentiation ◽  
Kidney Organoids

AbstractRecent advances in induced pluripotent stem cells (iPSCs), genome editing technologies and 3D organoid model systems highlight opportunities to develop new in vitro human disease models to serve drug discovery programs. An ideal disease model would accurately recapitulate the relevant disease phenotype and provide a scalable platform for drug and genetic screening studies. Kidney organoids offer a high cellular complexity that may provide greater insights than conventional single-cell type cell culture models. However, genetic manipulation of the kidney organoids requires prior generation of genetically modified clonal lines, which is a time and labor consuming procedure. Here, we present a methodology for direct differentiation of the CRISPR-targeted cell pools, using a doxycycline-inducible Cas9 expressing hiPSC line for high efficiency editing to eliminate the laborious clonal line generation steps. We demonstrate the versatile use of genetically engineered kidney organoids by targeting the autosomal dominant polycystic kidney disease (ADPKD) genes: PKD1 and PKD2. Direct differentiation of the respective knockout pool populations into kidney organoids resulted in the formation of cyst-like structures in the tubular compartment. Our findings demonstrated that we can achieve > 80% editing efficiency in the iPSC pool population which resulted in a reliable 3D organoid model of ADPKD. The described methodology may provide a platform for rapid target validation in the context of disease modeling.

Abstract 16185: Biomarker-based Heart Failure Risk Stratification in Patients With Atherosclerotic Cardiovascular Disease: Observations From HPS-3/TIMI-55-REVEAL

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
David Berg ◽  
Stephen D Wiviott ◽  
Eugene Braunwald ◽  
David A Morrow ◽  
Keyword(s):  
Heart Failure ◽  
Cardiovascular Disease ◽  
Myocardial Injury ◽  
Cox Regression ◽  
Troponin T ◽  
Risk Indicators ◽  
Atherosclerotic Cardiovascular Disease ◽  
Circulating Biomarkers ◽  
Clinical Risk ◽  
Hemodynamic Stress

Background: Circulating biomarkers reflecting pathways implicated in heart failure (HF) may improve HF risk assessment in patients with stable atherosclerotic cardiovascular disease (ASCVD). Hypothesis: We aimed to evaluate the performance of circulating biomarkers of hemodynamic stress, myocardial injury, and inflammation for the prediction of hospitalization for HF (HHF) in a large well-characterized cohort with stable ASCVD followed for a median of 4.1 years. Methods: HPS3/TIMI 55-REVEAL was a randomized, double-blind, placebo-controlled trial of the CETP inhibitor anacetrapib in patients with stable ASCVD. We performed a nested prospective biomarker study, measuring high-sensitivity troponin T (hsTnT), N-terminal pro-B-type natriuretic peptide (NT-proBNP), and growth differentiation factor-15 (GDF-15) (all Roche Diagnostics) at randomization (n=29,673). Hazard ratios were adjusted for covariates of a priori clinical relevance to HHF risk: age, prior HF, hypertension, diabetes mellitus, eGFR <60, body-mass index, and polyvascular disease. Discrimination was assessed using Harrell’s c-index. Results: A significant graded risk of HHF was observed with increasing deciles of hsTnT, NT-proBNP, and GDF-15 (p-trend <0.001 for each) ( Figure ). These associations remained significant after multivariable adjustment for clinical risk factors (p<0.001 for each). When added to a multivariable Cox regression model of clinical risk indicators (c-index 0.74), these 3 biomarkers significantly improved the prognostic performance of the model (c-index 0.85; p<0.001). There was no treatment interaction with anacetrapib. Conclusions: In patients with stable ASCVD, biomarkers of myocardial injury, hemodynamic stress, and inflammation provide incremental information for prediction of HHF. Future studies should address whether these patients are more likely to benefit from emerging HF preventive therapies.

scholarly journals Downregulation of nNOS and synthesis of PGs associated with endotoxin-induced delay in gastric emptying

2002 ◽  
Vol 283 (6) ◽  
pp. G1360-G1367 ◽  
Author(s):  
Sara Calatayud ◽  
Eugenia García-Zaragozá ◽  
Carlos Hernández ◽  
Elsa Quintana ◽  
Vicente Felipo ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Gastric Emptying ◽  
Methyl Ester ◽  
Intraperitoneal Injection ◽  
Delayed Gastric Emptying ◽  
Single Intraperitoneal Injection ◽  
Inos Inhibitor ◽  
Oxide Synthase ◽  
Experimental Group ◽  
Inducible Nos

A single intraperitoneal injection of endotoxin (40 μg/kg) significantly delayed gastric emptying of a solid nutrient meal. Blockade of nitric oxide synthase (NOS) with 30 mg/kg ip N G-nitro-l-arginine methyl ester or 20 mg/kg ip 7-nitroindazole [neuronal NOS (nNOS) inhibitor] significantly delayed gastric emptying in control animals but failed to modify gastric emptying in endotoxin-treated rats. Administration of 2.5, 5, and 10 mg/kg ip N 6-iminoethyl-l-lysine [inducible NOS (iNOS) inhibitor] had no effect in either experimental group. Indomethacin (5 mg/kg sc), NS-398 (cyclooxygenase-2 inhibitor; 10 mg/kg ip), and dexamethasone (10 mg/kg sc) but not quinacrine (20 mg/kg ip) significantly prevented delay in gastric emptying induced by endotoxin but failed to modify gastric emptying in vehicle-treated animals. Ca2+-dependent NOS activity in the antrum pylorus of the stomach was diminished by endotoxin, whereas Ca2+-independent NOS activity was not changed. In addition, decreased nNOS mRNA and protein were observed in the antrum pylorus of endotoxin-treated rats. Our results suggest that downregulation of nNOS in the antrum pylorus of the stomach and synthesis of prostaglandins mediate the delay in gastric emptying of a solid nutrient meal induced by endotoxin.

A single intraperitoneal injection of bovine fetuin-A attenuates bone resorption in a murine calvarial model of particle-induced osteolysis

Bone ◽  
2017 ◽  
Vol 105 ◽  
pp. 262-268 ◽  
Author(s):  
Heidrun Jablonski ◽  
Christina Polan ◽  
Christian Wedemeyer ◽  
Gero Hilken ◽  
Rüdiger Schlepper ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Intraperitoneal Injection ◽  
Fetuin A ◽  
Single Intraperitoneal Injection

Application of biomaterials to in vitro pluripotent stem cell disease modeling of the skeletal system

2016 ◽  
Vol 4 (20) ◽  
pp. 3482-3489 ◽  
Author(s):  
Giuliana E. Salazar-Noratto ◽  
Frank P. Barry ◽  
Robert E. Guldberg
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Genetic Manipulation ◽  
Embryonic Stem ◽  
Cell Disease ◽  
Skeletal System ◽  
System P ◽  
Induced Pluripotent

Disease-specific pluripotent stem cells can be derived through genetic manipulation of embryonic stem cells or by reprogramming somatic cells (induced pluripotent stem cells).

Neuroprotection by glucagon: role of gluconeogenesis

2011 ◽  
Vol 114 (1) ◽  
pp. 85-91 ◽  
Author(s):  
Rami Abu Fanne ◽  
Taher Nassar ◽  
Achinoam Mazuz ◽  
Otailah Waked ◽  
Samuel N. Heyman ◽  
...  
Keyword(s):  
Blood Glucose ◽  
Amino Acid ◽  
Intraperitoneal Injection ◽  
Neuroprotective Effect ◽  
Closed Head Injury ◽  
Lesion Size ◽  
Hepatic Uptake ◽  
Glucose Levels ◽  
Single Intraperitoneal Injection ◽  
Amino Acid Glutamate

Object The severity of neurological impairment following traumatic brain injury (TBI) is exacerbated by several endogenous processes, including hyperglycemia, hypotension, and the generation of glutamate. However, in addition to controlling hyperglycemia, insulin has pleiotropic effects on tissue metabolism, which include reducing the concentration of the neurotoxic amino acid glutamate, making it unclear whether insulin's beneficial effects are attributable to the establishment of euglycemia per se. In the present study, the authors asked if reducing glutamate via approaches that do not lower glucose levels would improve neurological outcome following TBI. Methods Glucagon activates gluconeogenesis by increasing the hepatic uptake of amino acids such as glutamate and facilitating their conversion to glucose. Glucagon was administered as a single intraperitoneal injection before or after closed head injury (CHI). Neurological function, brain histological features, blood glutamate and glucose levels, and CSF glutamate concentrations were measured. Results A single intraperitoneal injection of glucagon (25 μg) into mice 10 minutes before or after CHI reduced lesion size by about 60% (p < 0.0001) and accelerated neurological recovery. The neuroprotective effect of glucagon was related to gluconeogenesis by decreasing the concentration of the neuroexcitatory amino acid glutamate in the circulation from 207 ± 32.1 μmol/L in untreated mice to 101.11 ± 21.6 μmol/L in treated mice (p < 0.001); a similar effect occurred in the CSF. The neuroprotective effect of glucagon was seen notwithstanding the attendant increase in blood glucose, the final substrate of gluconeogenesis. Conclusions Glucagon exerts a marked neuroprotective effect post-TBI by decreasing CNS glutamate. Glucagon was beneficial despite increasing blood glucose. Favorable effects also occurred when glucagon was given prior to TBI, suggesting its involvement in the preconditioning process. Thus, glucagon may be of value in providing neuroprotection when administered after TBI or prior to certain neurosurgical or cardiac interventions in which the incidence of perioperative ischemia is high.

Duration of Availability of Tritiated Thymidine Following Intraperitoneal Injection

1968 ◽  
Vol 3 (1) ◽  
pp. 89-93
Author(s):  
W. K. BLENKINSOPP
Keyword(s):  
Cell Division ◽  
Intraperitoneal Injection ◽  
Deoxyribonucleic Acid ◽  
Direct Evidence ◽  
Tritiated Thymidine ◽  
Indirect Evidence ◽  
Acid Synthesis ◽  
Single Intraperitoneal Injection ◽  
Deoxyribonucleic Acid Synthesis

Much indirect evidence supports the assumption that tritiated thymidine does not label cells which enter the deoxyribonucleic acid synthesis phase (S) more than 1 h after injection. Direct evidence confirming this assumption was obtained by counting labelled epithelial nuclei in mice killed 1, 4 or 6 h after a single intraperitoneal injection of [3H]thymidine; colchicine was used to prevent the increase in number of labelled nuclei which would otherwise have occurred because of cell division. The proportion of cells labelled was the same at 1 h as at 4 or 6 h after injection of [3H]thymidine. Nuclei were regarded as labelled if they were overlaid by 4 grains or more; comparison of nuclear and background labelling indicated that nuclei overlaid by 3 grains or less represented background labelling.

Abstract 516: Chromosome 18p Deletion Inhibits Platelet Aggregation

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Andrew D Meyer ◽  
Anjana Raghunath ◽  
Patricia Heard ◽  
Jannine Cody
Keyword(s):  
Cardiovascular Disease ◽  
Platelet Aggregation ◽  
Platelet Function ◽  
Disease Modeling ◽  
Significant Risk ◽  
The United States ◽  
Gene Copy ◽  
Impedance Aggregometry ◽  
Significant Difference ◽  
Normal Controls

Inappropriate platelet function is a significant risk factor for cardiovascular disease, the leading cause of death in the United States. Although abnormal platelet function has a strong genetic component, very few human genes have been linked to platelet function. Mice with a homozygous deletion of EMILIN2 (Elastin Microfibril Interface Located Protein2) gene, located on Chromosome 18p, have a significant decrease in platelet function and clot formation. However, deletion or inactivation of only one copy of a gene is most relevant to human disease modeling. Our hypothesis is that blood samples from people with single 18p deletions that include EMILIN2 will have decreased platelet function compared to healthy individuals. We conducted a case-control study of nine adult individuals with chromosome 18p deletions matched with healthy men and women (n=20). Routine coagulation measurements were performed on a STAGO STA-R instrument. Platelet aggregation was measured with whole blood impedance aggregometry and Thromboelastography with PlateletMapping using the manufacturers’ protocols. There was no significant difference in platelet count, prothrombin time, partial thromboplastin time, d-dimer, or fibrinogen between individuals with a single 18p gene copy number and normal controls. However, platelet aggregation was impaired in individuals with 18p deletions compared to normal controls in response to collagen and arachidonic acid (ASPI), respectively ( p <0.0001, Figure 1). Moreover, Thromboelastography with PlateletMapping was decreased in individuals with 18p deletions compared to normal controls for ADP and ASPI, ( p <0.001). Individuals with one copy of 18p have decrease platelet function compared to normal controls. These results identify a novel human genetic loci linked to a specific phenotype of platelet function. Future will studies will determine if this gene can be used for diagnostic or therapeutics for cardiovascular disease.

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