Conditional deletion of Ccl2 in smooth muscle cells does not reduce early atherosclerosis in mice.

Background and aims: C-C motif chemokine ligand 2 (CCL2) is a pro-inflammatory chemokine important for monocyte recruitment to the arterial wall and atherosclerotic plaques. Global knockout of Ccl2 reduces plaque formation and macrophage content in mice, but the importance of different plaque cell types in mediating this effect has not been resolved. Smooth muscle cells (SMCs) can adopt a potentially pro-inflammatory function with expression of CCL2. The present study aimed to test the hypothesis that SMC-secreted CCL2 is involved in early atherogenesis in mice. Methods: SMC-restricted Cre recombinase was activated at 6 weeks of age in mice with homozygous floxed or wildtype Ccl2 alleles. Separate experiments in mice lacking the Cre recombinase transgene were conducted to control for genetic background effects. Hypercholesterolemia and atherosclerosis were induced by a tail vein injection of recombinant adeno-associated virus (rAAV) encoding proprotein convertase subtilisin/kexin type 9 (PCSK9) and a high-fat diet for 12 weeks. Results: Unexpectedly, mice with SMC-specific Ccl2 deletion developed higher levels of plasma cholesterol and larger atherosclerotic plaques with more macrophages compared with wild-type littermates. When total cholesterol levels were incorporated into the statistical analysis, none of the effects on plaque development between groups remained significant. Importantly, changes in plasma cholesterol and atherosclerosis remained in mice lacking Cre recombinase indicating that they were not caused by SMC-specific CCL2 deletion but by effects of the floxed allele or passenger genes. Conclusions: SMC-specific deficiency of Ccl2 does not significantly affect early plaque development in hypercholesterolemic mice.

Severe arterial injury heals with a complex clonal structure involving a large fraction of surviving smooth muscle cells.

BACKGROUND AND AIMS: Smooth muscle cell (SMC) lineage cells in atherosclerosis and flow cessation-induced neointima are oligoclonal, being recruited from a tiny fraction of medial SMCs that modulate and proliferate. The present study aimed to investigate the clonal structure of SMC lineage cells healing more severe arterial injury. METHODS: Arterial injury (wire, stretch, and partial ligation) was inflicted on the right carotid artery in mice with homozygous, SMC-restricted, stochastically recombining reporter transgenes that produced mosaic expression of 10 distinguishable fluorescent phenotypes for clonal tracking. Healed arteries and contra-lateral controls were analyzed after 3 weeks. Additional analysis of cell death and proliferation after injury was performed in wildtype mice. RESULTS: The total number of SMC lineage cells in healed arteries was comparable to normal arteries but comprised significantly fewer fluorescent phenotypes. The population had a complex, intermixed, clonal structure. By statistical analysis of expected versus observed fractions of fluorescent phenotypes and visual inspection of coherent groups of same-colored cells, we concluded that >98% of SMC lineage cells in healed arteries belonged to a detectable clone, indicating that nearly all surviving SMCs after severe injury at some point undergo proliferation. This was consistent with serial observations in the first week after injury, which showed severe loss of medial cells followed by widespread proliferation. CONCLUSIONS: After severe arterial injury, many surviving SMCs proliferate to repair the media and form a neointima. This indicates that the fraction of medial SMCs that are mobilized to repair arteries increases with the level of injury.

Impact of Administration Time and Kv7 Subchannels on the Cardioprotective Efficacy of Kv7 Channel Inhibition.

PURPOSE: The mechanism of cardioprotection by Kv7.1-5 (KCNQ1-5) channels inhibition by XE991 is unclear. We examined the impact of administration time on the cardioprotective efficacy of XE991, the involvement of key pro-survival kinases, and the importance of the Kv7 subchannels. METHODS: Isolated perfused rat hearts were divided into five groups: 1) vehicle, 2) pre-, 3) post- or 4) pre- and post-ischemic administration of XE991 or 5) chromanol 293B (Kv7.1 inhibitor) followed by infarct size quantification. HL-1 cells undergoing simulated ischemia/reperfusion were exposed to either a) vehicle, b) pre-, c) per-, d) post-ischemic administration of XE991 or pre-, per- and post-ischemic administration of e) XE991, f) Chromanol 293B or g) HMR1556 (Kv7.1 inhibitor). HL-1 cell injury was evaluated by propidium iodide/Hoechst staining. Pro-survival kinase activation of Akt, Erk and STAT3 in XE991-mediated HL-1 cell protection was evaluated using phosphokinase inhibitors. Kv7 subtype expression was examined by RT-PCR and qPCR. RESULTS: XE991, but not Chromanol 293B, reduced infarct size and improved hemodynamic recovery in all isolated heart groups. XE991 protected HL-1 cells when administered during simulated ischemia. Minor activation of the survival kinases was observed in cells exposed to XE991 but pharmacological inhibition of kinase activation did not reduce XE991-mediated protection. Kv7 subchannels 1-5 were all present in rat hearts but predominately Kv7.1 and Kv7.4 were present in HL-1 cells and selective Kv7.1 did not reduce ischemia/reperfusion injury. CONCLUSION: The cardioprotective efficacy of XE991 seems to depend on its presence during ischemia and early reperfusion and do not rely on RISK (p-Akt and p-Erk) and SAFE (p-STAT3) pathway activation. The protective effect of XE991 seems mainly mediated through the Kv7.4 subchannel.

Generation and characterization of a novel knockin minipig model of Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disorder for which no cure exists. The disease is characterized by premature aging and inevitable death in adolescence due to cardiovascular complications. Most HGPS patients carry a heterozygous de novo LMNA c.1824C > T mutation, which provokes the expression of a dominant-negative mutant protein called progerin. Therapies proven effective in HGPS-like mouse models have yielded only modest benefit in HGPS clinical trials. To overcome the gap between HGPS mouse models and patients, we have generated by CRISPR-Cas9 gene editing the first large animal model for HGPS, a knockin heterozygous LMNA c.1824C > T Yucatan minipig. Like HGPS patients, HGPS minipigs endogenously co-express progerin and normal lamin A/C, and exhibit severe growth retardation, lipodystrophy, skin and bone alterations, cardiovascular disease, and die around puberty. Remarkably, the HGPS minipigs recapitulate critical cardiovascular alterations seen in patients, such as left ventricular diastolic dysfunction, altered cardiac electrical activity, and loss of vascular smooth muscle cells. Our analysis also revealed reduced myocardial perfusion due to microvascular damage and myocardial interstitial fibrosis, previously undescribed readouts potentially useful for monitoring disease progression in patients. The HGPS minipigs provide an appropriate preclinical model in which to test human-size interventional devices and optimize candidate therapies before advancing to clinical trials, thus accelerating the development of effective applications for HGPS patients.

Differences in Hypercholesterolemia and Atherogenesis Induced by Common Androgen Deprivation Therapies in Male Mice.

BACKGROUND: Treatment of prostate cancer often involves androgen deprivation therapy (ADT) by gonadotropin-releasing hormone (GnRH) receptor agonists, GnRH receptor antagonists, or orchiectomy. ADT may increase the rate of cardiovascular disease events, but recent clinical studies suggested that not all means of ADT carry the same risk, raising the possibility of non-testosterone-mediated effects of different forms of ADT on atherosclerosis. Here we compared effects of ADT on atherosclerosis in intact and orchiectomized Apoe-deficient mice. METHODS AND RESULTS: Chow-fed Apoe-deficient mice were allocated to orchiectomy and/or monthly injections with the GnRH receptor agonist leuprolide or the GnRH receptor antagonist degarelix. Atherosclerosis was quantified at 26 weeks of age in the aortic arch by en face examination and in the aortic root by histology. In intact Apoe-deficient mice, all types of ADT reduced testosterone production to castration levels. Although hypercholesterolemia was accentuated in leuprolide-treated mice, the amount and composition of atherosclerosis was not different between the different types of ADT. In orchiectomized Apoe-deficient mice, leuprolide, but not degarelix, augmented hypercholesterolemia, changed body, thymus, and spleen weights, and increased atherosclerosis in the aortic root. No direct effects of the drugs were detectable on cytokine secretion from murine bone marrow-derived macrophages or on splenocyte proliferation. CONCLUSIONS: No differences in the development of atherosclerosis were detected among groups of intact Apoe-deficient mice treated with different types of ADT. A pro-atherogenic, possibly cholesterol-mediated, effect of leuprolide was seen in orchiectomized mice that might be relevant for understanding the potential cardiovascular risk associated with GnRH agonist-based ADT.

Enhanced genome editing in mammalian cells with a modified dual-fluorescent surrogate system.

Programmable DNA nucleases such as TALENs and CRISPR/Cas9 are emerging as powerful tools for genome editing. Dual-fluorescent surrogate systems have been demonstrated by several studies to recapitulate DNA nuclease activity and enrich for genetically edited cells. In this study, we created a single-strand annealing-directed, dual-fluorescent surrogate reporter system, referred to as C-Check. We opted for the Golden Gate Cloning strategy to simplify C-Check construction. To demonstrate the utility of the C-Check system, we used the C-Check in combination with TALENs or CRISPR/Cas9 in different scenarios of gene editing experiments. First, we disrupted the endogenous pIAPP gene (3.0 % efficiency) by C-Check-validated TALENs in primary porcine fibroblasts (PPFs). Next, we achieved gene-editing efficiencies of 9.0-20.3 and 4.9 % when performing single- and double-gene targeting (MAPT and SORL1), respectively, in PPFs using C-Check-validated CRISPR/Cas9 vectors. Third, fluorescent tagging of endogenous genes (MYH6 and COL2A1, up to 10.0 % frequency) was achieved in human fibroblasts with C-Check-validated CRISPR/Cas9 vectors. We further demonstrated that the C-Check system could be applied to enrich for IGF1R null HEK293T cells and CBX5 null MCF-7 cells with frequencies of nearly 100.0 and 86.9 %, respectively. Most importantly, we further showed that the C-Check system is compatible with multiplexing and for studying CRISPR/Cas9 sgRNA specificity. The C-Check system may serve as an alternative dual-fluorescent surrogate tool for measuring DNA nuclease activity and enrichment of gene-edited cells, and may thereby aid in streamlining programmable DNA nuclease-mediated genome editing and biological research.

Absence of an intron splicing silencer in porcine Smn1 intron 7 confers immunity to the exon skipping mutation in human SMN2.

Spinal Muscular Atrophy is caused by homozygous loss of SMN1. All patients retain at least one copy of SMN2 which produces an identical protein but at lower levels due to a silent mutation in exon 7 which results in predominant exclusion of the exon. Therapies targeting the splicing of SMN2 exon 7 have been in development for several years, and their efficacy has been measured using either in vitro cellular assays or in vivo small animal models such as mice. In this study we evaluated the potential for constructing a mini-pig animal model by introducing minimal changes in the endogenous porcine Smn1 gene to maintain the native genomic structure and regulation. We found that while a Smn2-like mutation can be introduced in the porcine Smn1 gene and can diminish the function of the ESE, it would not recapitulate the splicing pattern seen in human SMN2 due to absence of a functional ISS immediately downstream of exon 7. We investigated the ISS region and show here that the porcine ISS is inactive due to disruption of a proximal hnRNP A1 binding site, while a distal hnRNP A1 binding site remains functional but is unable to maintain the functionality of the ISS as a whole.

Efficient construction of rAAV-based gene targeting vectors by Golden Gate cloning.

The recombinant adeno-associated virus (rAAV) has proven to be an efficient and attractive tool for targeted genome engineering. Here we present a novel method employing the Golden Gate cloning strategy for fast and efficient construction of rAAV-based gene knockout or single-nucleotide knockin vectors. Two vectors, pGolden-Neo and pGolden-Hyg, were generated as common assembling modules to confer antibiotic resistance to the targeting vector. To validate the method, we then generated two rAAV-based targeting vectors: pAAV-pTP53-KO and pAAV-hTau(P301L)-KI. Furthermore, we generated a pGolden-AAV plasmid that allows one-step generation of an rAAV-based targeting vector. Our new methodology for rAAV targeting vector assembly is efficient, accurate, time-saving, and cost-effective.

Targeted genome editing by recombinant adeno-associated virus (rAAV) vectors for generating genetically modified pigs.

Recombinant adeno-associated virus (rAAV) vectors have been extensively used for experimental gene therapy of inherited human diseases. Several advantages, such as simple vector construction, high targeting frequency by homologous recombination, and applicability to many cell types, make rAAV an attractive approach for targeted genome editing. Combined with cloning by somatic cell nuclear transfer (SCNT), this technology has recently been successfully adapted to generate gene-targeted pigs as models for cystic fibrosis, hereditary tyrosinemia type 1, and breast cancer. This review summarizes the development of rAAV for targeted genome editing in mammalian cells and provides strategies for enhancing the rAAV-mediated targeting frequency by homologous recombination. We discuss current development and application of the rAAV vectors for targeted genome editing in porcine primary fibroblasts, which are subsequently used as donor cells for SCNT to generate cloned genetically designed pigs and provide positive perspectives for the generation of gene-targeted pigs with rAAV in the future.

Genetically modified pigs for biomedical research.

During the last two decades, pigs have been used to develop some of the most important large animal models for biomedical research. Advances in pig genome research, genetic modification (GM) of primary pig cells and pig cloning by nuclear transfer, have facilitated the generation of GM pigs for xenotransplantation and various human diseases. This review summarizes the key technologies used for generating GM pigs, including pronuclear microinjection, sperm-mediated gene transfer, somatic cell nuclear transfer by traditional cloning, and somatic cell nuclear transfer by handmade cloning. Broadly used genetic engineering tools for porcine cells are also discussed. We also summarize the GM pig models that have been generated for xenotransplantation and human disease processes, including neurodegenerative diseases, cardiovascular diseases, eye diseases, bone diseases, cancers and epidermal skin diseases, diabetes mellitus, cystic fibrosis, and inherited metabolic diseases. Thus, this review provides an overview of the progress in GM pig research over the last two decades and perspectives for future development.

Pig gene knockout by rAAV-mediated homologous recombination: comparison of BRCA1 gene knockout efficiency in Yucatan and Göttingen fibroblasts with slightly different target sequences.

In this study, we compared the gene targeting efficiencies of two rAAV-BRCA1 KO targeting constructs in Yucatan and Göttingen minipig fibroblasts. The homology arms of the constructs consisted exclusively of exonic sequences amplified by PCR from Yucatan genomic DNA. The sequences were identical to those of the reference porcine genome of a Duroc sow (Ensembl Susscrofa 9) and the BRCA1 gene of the Landrace breed (NCBI acc. no. AB271921). Surprisingly, we found that the very efficient gene targeting observed for Yucatan fibroblasts (35% targeting efficiency) was completely absent using either of the two constructs in Göttingen fibroblasts. Sequencing of the relevant BRCA1 exon 11 region (~2 kb) in the Göttingen minipig revealed three single nucleotide differences in the sequence targeted by the left homology arm of the construct (0.3% of the bases) and three or seven in the two right homology regions (0.3 or 0.7% of the bases, respectively). Construction of a novel rAAV-BRCA1 targeting vector based on the Göttingen genomic DNA sequence re-established gene targeting although the efficiency was somewhat lower than that observed for Yucatan fibroblasts. These BRCA1 KO Göttingen fibroblast clones have been used as nuclear donor cells for somatic cell nuclear transfer to generate a Göttingen BRCA1 KO pig model as previously done with the Yucatan breed. The present study illustrates that even a few mismatches present in the homology arms of an efficient rAAV-targeting construct can completely abolish gene targeting by homologous recombination emphasizing the importance of using isogenic DNA even for creating targeting constructs consisting of exon sequences only.

Comparison of gene expression and genome-wide DNA methylation profiling between phenotypically normal cloned pigs and conventionally bred controls.

Animal breeding via Somatic Cell Nuclear Transfer (SCNT) has enormous potential in agriculture and biomedicine. However, concerns about whether SCNT animals are as healthy or epigenetically normal as conventionally bred ones are raised as the efficiency of cloning by SCNT is much lower than natural breeding or In-vitro fertilization (IVF). Thus, we have conducted a genome-wide gene expression and DNA methylation profiling between phenotypically normal cloned pigs and control pigs in two tissues (muscle and liver), using Affymetrix Porcine expression array as well as modified methylation-specific digital karyotyping (MMSDK) and Solexa sequencing technology. Typical tissue-specific differences with respect to both gene expression and DNA methylation were observed in muscle and liver from cloned as well as control pigs. Gene expression profiles were highly similar between cloned pigs and controls, though a small set of genes showed altered expression. Cloned pigs presented a more different pattern of DNA methylation in unique sequences in both tissues. Especially a small set of genomic sites had different DNA methylation status with a trend towards slightly increased methylation levels in cloned pigs. Molecular network analysis of the genes that contained such differential methylation loci revealed a significant network related to tissue development. In conclusion, our study showed that phenotypically normal cloned pigs were highly similar with normal breeding pigs in their gene expression, but moderate alteration in DNA methylation aspects still exists, especially in certain unique genomic regions.

High efficiency of BRCA1 knockout using rAAV-mediated gene targeting: developing a pig model for breast cancer.

Germline inactivating mutations of the breast cancer associated gene 1 (BRCA1) predispose to breast cancer and account for most cases of familiar breast and/or ovarian cancer. The pig is an excellent model for medical research as well as testing of new methods and drugs for disease prevention and treatment. We have generated cloned BRCA1 knockout (KO) Yucatan miniature piglets by targeting exon 11 using recombinant adeno-associated virus (rAAV)-mediated gene targeting and somatic cell nuclear transfer by Handmade Cloning (HMC). We found a very high targeting rate of rAAV-mediated BRCA1 KO. Approximately 35% of the selected cells were BRCA1 targeted. One BRCA1 KO cell clone (5D1), identified by PCR and Southern blot, was used as nuclear donor for HMC. Reconstructed embryos were transferred to three recipient sows which gave birth to 8 piglets in total. Genotyping identified seven piglets as BRCA1 heterozygotes (BRCA1(+/∆11)), and one as wild type. The BRCA1 expression was decreased at the mRNA level in BRCA1(+/∆11) fibroblasts. However, all BRCA1(+/∆11) piglets died within 18 days after birth. The causes of perinatal mortality remain unclear. Possible explanations may include a combination of the BRCA1 haploinsufficiency, problems of epigenetic reprogramming, presence of the marker gene, single cell clone effects, and/or the special genetic background of the minipigs.