Cre-loxP Recombination Research Articles

Mammary alveolar epithelial cells convert to brown adipocytes in post-lactating mice.

During pregnancy and lactation, subcutaneous white adipocytes in the mouse mammary gland transdifferentiate reversibly to milk-secreting epithelial cells. In this study, we demonstrate by transmission electron microscopy that in the post-lactating mammary gland interscapular multilocular adipocytes found close to the mammary alveoli contain milk protein granules. Use of the Cre-loxP recombination system allowed showing that the involuting mammary gland of whey acidic protein-Cre/R26R mice, whose secretory alveolar cells express the lacZ gene during pregnancy, contains some X-Gal-stained and uncoupling protein 1-positive interscapular multilocular adipocytes. These data suggest that during mammary gland involution some milk-secreting epithelial cells in the anterior subcutaneous depot may transdifferentiate to brown adipocytes, highlighting a hitherto unappreciated feature of mouse adipose organ plasticity.

The role of platelet and endothelial GARP in thrombosis and hemostasis.

BackgroundGlycoprotein-A Repetitions Predominant protein (GARP or LRRC32) is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish.ObjectivesTo evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice.MethodsPlatelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice.ResultsPlatelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin αIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected.ConclusionsEvidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice.

Preparing Mate-Paired Illumina Libraries Using Cre Recombinase.

Large insert mate pair reads have been used in de novo assembly and discovery of structural variants. We developed a new approach, Cre-LoxP inverse PCR paired end (CLIP-PE), which exploits the advantages of (1) Cre-LoxP recombination system to efficiently circularize large DNA fragments, (2) inverse PCR to enrich for the desired products that contain both ends of the large DNA fragments, and (3) use of restriction enzymes to introduce a recognizable junction site between ligated fragment ends. We have successfully created CLIP-PE libraries of up to 22kb jumping pairs and demonstrated their ability to improve genome assemblies. The CLIP-PE methodology can be implemented with existing and future next-generation sequencing platforms.

A photoactivatable Cre-loxP recombination system for optogenetic genome engineering.

Genome engineering techniques represented by the Cre-loxP recombination system have been used extensively for biomedical research. However, powerful and useful techniques for genome engineering that have high spatiotemporal precision remain elusive. Here we develop a highly efficient photoactivatable Cre recombinase (PA-Cre) to optogenetically control genome engineering in vivo. PA-Cre is based on the reassembly of split Cre fragments by light-inducible dimerization of the Magnet system. PA-Cre enables sharp induction (up to 320-fold) of DNA recombination and is efficiently activated even by low-intensity illumination (∼0.04 W m-2) or short periods of pulsed illumination (∼30 s). We demonstrate that PA-Cre allows for efficient DNA recombination in an internal organ of living mice through noninvasive external illumination using a LED light source. The present PA-Cre provides a powerful tool to greatly facilitate optogenetic genome engineering in vivo.

Muscle expression of a malonyl-CoA-insensitive carnitine palmitoyltransferase-1 protects mice against high-fat/high-sucrose diet-induced insulin resistance.

Impaired skeletal muscle mitochondrial fatty acid oxidation (mFAO) has been implicated in the etiology of insulin resistance. Carnitine palmitoyltransferase-1 (CPT1) is a key regulatory enzyme of mFAO whose activity is inhibited by malonyl-CoA, a lipogenic intermediate. Whereas increasing CPT1 activity in vitro has been shown to exert a protective effect against lipid-induced insulin resistance in skeletal muscle cells, only a few studies have addressed this issue in vivo. We thus examined whether a direct modulation of muscle CPT1/malonyl-CoA partnership is detrimental or beneficial for insulin sensitivity in the context of diet-induced obesity. By using a Cre-LoxP recombination approach, we generated mice with skeletal muscle-specific and inducible expression of a mutated CPT1 form (CPT1mt) that is active but insensitive to malonyl-CoA inhibition. When fed control chow, homozygous CPT1mt transgenic (dbTg) mice exhibited decreased CPT1 sensitivity to malonyl-CoA inhibition in isolated muscle mitochondria, which was sufficient to substantially increase ex vivo muscle mFAO capacity and whole body fatty acid utilization in vivo. Moreover, dbTg mice were less prone to high-fat/high-sucrose (HFHS) diet-induced insulin resistance and muscle lipotoxicity despite similar body weight gain, adiposity, and muscle malonyl-CoA content. Interestingly, these CPT1mt-protective effects in dbTg-HFHS mice were associated with preserved muscle insulin signaling, increased muscle glycogen content, and upregulation of key genes involved in muscle glucose metabolism. These beneficial effects of muscle CPT1mt expression suggest that a direct modulation of the malonyl-CoA/CPT1 partnership in skeletal muscle could represent a potential strategy to prevent obesity-induced insulin resistance.

Dicer ablation in osteoblasts by Runx2 driven cre-loxP recombination affects bone integrity, but not glucocorticoid-induced suppression of bone formation.

Glucocorticoid-induced osteoporosis (GIO) is one of the major side effects of long-term glucocorticoid (GC) therapy mediated mainly via the suppression of bone formation and osteoblast differentiation independently of GC receptor (GR) dimerization. Since microRNAs play a critical role in osteoblast differentiation processes, we investigated the role of Dicer dependent microRNAs in the GC-induced suppression of osteoblast differentiation. MicroRNA sequencing of dexamethasone-treated wild-type and GR dimer-deficient mesenchymal stromal cells revealed GC-controlled miRNA expression in a GR dimer-dependent and GR dimer-independent manner. To determine the functional relevance of mature miRNAs in GC-induced osteoblast suppression, mice with an osteoblast-specific deletion of Dicer (DicerRunx2Cre) were exposed to glucocorticoids. In vitro generated Dicer-deficient osteoblasts were treated with dexamethasone and analyzed for proliferation, differentiation and mineralization capacity. In vivo, abrogation of Dicer-dependent miRNA biogenesis in osteoblasts led to growth retardation and impaired bone formation. However, subjecting these mice to GIO showed that bone formation was similar reduced in DicerRunx2Cre mice and littermate control mice upon GC treatment. In line, differentiation of Dicer deficient osteoblasts was suppressed to the same extent as wild type cells by GC treatment. Therefore, Dicer-dependent small RNA biogenesis in osteoblasts plays only a minor role in the pathogenesis of GC-induced inhibition of bone formation.

Leukocyte Calpain Deficiency Reduces Angiotensin II-Induced Inflammation and Atherosclerosis But Not Abdominal Aortic Aneurysms in Mice.

Angiotensin II (AngII) infusion profoundly increases activity of calpains, calcium-dependent neutral cysteine proteases, in mice. Pharmacological inhibition of calpains attenuates AngII-induced aortic medial macrophage accumulation, atherosclerosis, and abdominal aortic aneurysm in mice. However, the precise functional contribution of leukocyte-derived calpains in AngII-induced vascular pathologies has not been determined. The purpose of this study was to determine whether calpains expressed in bone marrow (BM)-derived cells contribute to AngII-induced atherosclerosis and aortic aneurysms in hypercholesterolemic mice. To study whether leukocyte calpains contributed to AngII-induced aortic pathologies, irradiated male low-density lipoprotein receptor(-/-) mice were repopulated with BM-derived cells that were either wild-type or overexpressed calpastatin, the endogenous inhibitor of calpains. Mice were fed a fat-enriched diet and infused with AngII (1000 ng/kg per minute) for 4 weeks. Overexpression of calpastatin in BM-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches, but had no effect on aneurysm formation. Using either BM-derived cells from calpain-1-deficient mice or mice with leukocyte-specific calpain-2 deficiency generated using cre-loxP recombination technology, further studies demonstrated that independent deficiency of either calpain-1 or -2 in leukocytes modestly attenuated AngII-induced atherosclerosis. Calpastatin overexpression significantly attenuated AngII-induced inflammatory responses in macrophages and spleen. Furthermore, calpain inhibition suppressed migration and adhesion of macrophages to endothelial cells in vitro. Calpain inhibition also significantly decreased hypercholesterolemia-induced atherosclerosis in the absence of AngII. The present study demonstrates a pivotal role for BM-derived calpains in mediating AngII-induced atherosclerosis by influencing macrophage function.

Cre-Lox Neurogenetics: 20 Years of Versatile Applications in Brain Research and Counting….

Defining and manipulating specific neurons in the brain has garnered enormous interest in recent years, because such an approach is now widely recognized as crucial for deepening our understanding of how the brain works. When I started exploring the Cre-loxP recombination for brain research in the early 1990s, it was written off as a dead-end project by a young fool. Yet over the past 20 years, Cre-lox recombination-mediated neurogenetics has emerged as one of the most powerful and versatile technology platforms for cell-specific gene knockouts, transgenic overexpression, Brainbow imaging, neural pathway tracing with retrovirus and CLARITY, chemical genetics, and optogenetics. Its popularity and greater utility in neuroscience research is also largely thanks to the NIH’s bold Blueprint for Neuroscience Research Initiative to launch several Cre-driver resource projects, as well as individual laboratories and private research organizations. With newly-discovered, genetically-encoded molecules that are capable of responding to sonar and magnetic stimulation, for sonogenetics or magnetogenetics, respectively, or detecting rapid voltage changes in neurons, Cre-lox neurogenetics will continue to aid brain research for years to come.

Engineering super mycovirus donor strains of chestnut blight fungus by systematic disruption of multilocus vic genes

Transmission of mycoviruses that attenuate virulence (hypovirulence) of pathogenic fungi is restricted by allorecognition systems operating in their fungal hosts. We report the use of systematic molecular gene disruption and classical genetics for engineering fungal hosts with superior virus transmission capabilities. Four of five diallelic virus-restricting allorecognition [vegetative incompatibility (vic)] loci were disrupted in the chestnut blight fungus Cryphonectria parasitica using an adapted Cre-loxP recombination system that allowed excision and recycling of selectable marker genes (SMGs). SMG-free, quadruple vic mutant strains representing both allelic backgrounds of the remaining vic locus were then produced through mating. In combination, these super donor strains were able to transmit hypoviruses to strains that were heteroallelic at one or all of the virus-restricting vic loci. These results demonstrate the feasibility of modulating allorecognition to engineer pathogenic fungi for more efficient transmission of virulence-attenuating mycoviruses and enhanced biological control potential.

Development of a conditional liver tumor model by mifepristone-inducible Cre recombination to control oncogenic kras V12 expression in transgenic zebrafish.

Here we report a new transgenic expression system by combination of liver-specific expression, mifepristone induction and Cre-loxP recombination to conditionally control the expression of oncogenic krasV12. This transgenic system allowed expression of krasV12 specifically in the liver by a brief exposure of mifepristone to induce permanent genomic recombination mediated by the Cre-loxP system. We found that liver tumors were generally induced from multiple foci due to incomplete Cre-loxP recombination, thus mimicking naturally occurring human tumors resulting from one or a few mutated cells and clonal proliferation to form nodules. Similar to our earlier studies by both constitutive and inducible expression of the krasV12 oncogene, hepatocellular carcinoma (HCC) is the main type of liver tumor induced by krasV12 expression. Moreover, mixed tumors with hepatocellular adenoma and hepatoblastoma (HB) were also frequently observed. Molecular analyses also indicated similar increase of phosphorylated ERK1/2 in all types of liver tumors, but nuclear localization of β–catenin, a sign of malignant transformation, was found only in HCC and HB. Taken together, our new transgenic system reported in this study allows transgenic krasV12 expression specifically in the zebrafish liver only by a brief exposure of mifepristone to induce permanent genomic recombination mediated by the Cre-loxP system.

Generation of CRISPR/Cas9-mediated bicistronic knock-in ins1-cre driver mice.

In the present study, we generated novel cre driver mice for gene manipulation in pancreatic β cells. Using the CRISPR/Cas9 system, stop codon sequences of Ins1 were targeted for insertion of cre, including 2A sequences. A founder of C57BL/6J-Ins1em1 (cre) Utr strain was produced from an oocyte injected with pX330 containing the sequences encoding gRNA and Cas9 and a DNA donor plasmid carrying 2A-cre. (R26GRR x C57BL/6J-Ins1em1 (cre) Utr) F1 mice were histologically characterized for cre-loxP recombination in the embryonic and adult stages; cre-loxP recombination was observed in all pancreatic islets examined in which almost all insulin-positive cells showed tdsRed fluorescence, suggesting β cell-specific recombination. Furthermore, there were no significant differences in results of glucose tolerance test among genotypes (homo/hetero/wild). Taken together, these observations indicated that C57BL/6J-Ins1em1 (cre) Utr is useful for studies of glucose metabolism and the strategy of bicistronic cre knock-in using the CRISPR/Cas9 system could be useful for production of cre driver mice.

Editorial: Application of Genetically Encoded Indicators to Mammalian Central Nervous System.

Genetically encoded indicators are fluorescent proteins engineered to change their optical properties and report the dynamics of intracellular molecules or voltage. Encoded in a relatively compact size of DNA (typically 2–3 kilobase pairs), they are readily compatible with standard gene delivery techniques, including viral vectors and transgenesis. They can be thus expressed in specific types of cells with the use of cell type-specific promoters and/or Cre-loxP recombination system, which makes them beneficial for disentangling the complex mammalian neural circuitry. Another unique advantage of genetically encoded indicators is that they can be stably expressed for a long period, which allows chronic tracking of the same neuronal ensemble by longitudinal imaging, and offers unprecedented opportunities to investigate the neural basis of development, learning, and disease.

Blood-testis barrier and Sertoli cell function: lessons from SCCx43KO mice.

The gap junction protein connexin43 (CX43) plays a vital role in mammalian spermatogenesis by allowing for direct cytoplasmic communication between neighbouring testicular cells. In addition, different publications suggest that CX43 in Sertoli cells (SC) might be important for blood-testis barrier (BTB) formation and BTB homeostasis. Thus, through the use of the Cre-LoxP recombination system, a transgenic mouse line was developed in which only SC are deficient of the gap junction protein, alpha 1 (Gja1) gene. Gja1 codes for the protein CX43. This transgenic mouse line has been commonly defined as the SC specific CX43 knockout (SCCx43KO) mouse line. Within the seminiferous tubule, SC aid in spermatogenesis by nurturing germ cells and help them to proliferate and mature. Owing to the absence of CX43 within the SC, homozygous KO mice are infertile, have reduced testis size, and mainly exhibit spermatogenesis arrest at the level of spermatogonia, seminiferous tubules containing only SC (SC-only syndrome) and intratubular SC-clusters. Although the SC specific KO of CX43 does not seem to have an adverse effect on BTB integrity, CX43 influences BTB composition as the expression pattern of different BTB proteins (like OCCLUDIN, β-CATENIN, N-CADHERIN, and CLAUDIN11) is altered in mutant males. The supposed roles of CX43 in dynamic BTB regulation, BTB assembly and/or disassembly and its possible interaction with other junctional proteins composing this unique barrier are discussed. Data collectively indicate that CX43 might represent an important regulator of dynamic BTB formation, composition and function.

Autistic-Like Traits and Cerebellar Dysfunction in Purkinje Cell PTEN Knock-Out Mice.

Autism spectrum disorders (ASDs) are neurodevelopmental disorders characterized by impaired social interaction, isolated areas of interest, and insistence on sameness. Mutations in Phosphatase and tensin homolog missing on chromosome 10 (PTEN) have been reported in individuals with ASDs. Recent evidence highlights a crucial role of the cerebellum in the etiopathogenesis of ASDs. In the present study we analyzed the specific contribution of cerebellar Purkinje cell (PC) PTEN loss to these disorders. Using the Cre-loxP recombination system, we generated conditional knockout mice in which PTEN inactivation was induced specifically in PCs. We investigated PC morphology and physiology as well as sociability, repetitive behavior, motor learning, and cognitive inflexibility of adult PC PTEN-mutant mice. Loss of PTEN in PCs results in autistic-like traits, including impaired sociability, repetitive behavior and deficits in motor learning. Mutant PCs appear hypertrophic and show structural abnormalities in dendrites and axons, decreased excitability, disrupted parallel fiber and climbing fiber synapses and late-onset cell death. Our results unveil new roles of PTEN in PC function and provide the first evidence of a link between the loss of PTEN in PCs and the genesis of ASD-like traits.

Low levels of Survival Motor Neuron protein are sufficient for normal muscle function in the SMNΔ7 mouse model of SMA.

Spinal Muscular Atrophy (SMA) is an autosomal recessive disorder characterized by loss of lower motor neurons. SMA is caused by deletion or mutation of the Survival Motor Neuron 1 (SMN1) gene and retention of the SMN2 gene. The loss of SMN1 results in reduced levels of the SMN protein. SMN levels appear to be particularly important in motor neurons; however SMN levels above that produced by two copies of SMN2 have been suggested to be important in muscle. Studying the spatial requirement of SMN is important in both understanding how SMN deficiency causes SMA and in the development of effective therapies. Using Myf5-Cre, a muscle-specific Cre driver, and the Cre-loxP recombination system, we deleted mouse Smn in the muscle of mice with SMN2 and SMNΔ7 transgenes in the background, thus providing low level of SMN in the muscle. As a reciprocal experiment, we restored normal levels of SMN in the muscle with low SMN levels in all other tissues. We observed that decreasing SMN in the muscle has no phenotypic effect. This was corroborated by muscle physiology studies with twitch force, tetanic and eccentric contraction all being normal. In addition, electrocardiogram and muscle fiber size distribution were also normal. Replacement of Smn in muscle did not rescue SMA mice. Thus the muscle does not appear to require high levels of SMN above what is produced by two copies of SMN2 (and SMNΔ7).

Abstract 822: Genetically engineered mouse models of catastrophic pancreatic ductal adenocarcinoma

Abstract Carcinogenesis of pancreatic ductal adenocarcinoma (PDAC) has been well studied using genetically engineered mouse (GEM) models with abnormalities in activation of both RAS and E2 factor (E2F)-which is associated with the Rb-E2F pathway, and dysfunction of p53-which is associated with the transformed 3T3 cell double minute 2 (MDM2)-p53 pathway. Catastrophic progression of the malignancy is one of unique manifestation, which has not been recapitulated in the GEMs to study underlined mechanisms, while we have recently reported a new GEM developing PDAC to die in 3 weeks after birth due to the catastrophic progression [Yamaguchi T. et al, J Pathol. 2014; 234(2):228-138]. The GEM had the specific expression of SV40 tsA58 large T antigen (tsTAg) to inhibit p53 and Rb family molecules (Rb1, Rbl1 and Rbl2) on pancreas with the expression of oncogenic Kras (KrasG12D) by Pdx1 promoter-mediated Cre-loxP recombination. In this study, we sought to determine if the induced tsTAg on pancreatic epithelial cells with constitutive KrasG12D expression are enough to develop PDAC and catastrophic manifestation, by the generation of a new GEM based on Cre-loxP recombination and Tet-On system. In the GEM, KrasG12D constitutively expresses on pancreatic epithelial cells after the Cre-loxP recombination under the control Pdx1 promoter, while tsTAg expression can be regulated by Tet-On system after the recombination by Pdx1 promoter. Any of the mice did not developed PDAC within 6 month, but all of the mice developed PDAC by Dox administration in within 1 week and died by the progression of PDAC within 3 weeks after the start.The rapid progression and development of PDAC appeared to be associated with the stronger tsTAg expression depending on reverse tetracycline trans-activator. These results demonstrated that KrasG12D driven PDAC carcinogenesis was promoted by the presence of tsTAg expression, resulting in production of PDAC cells with aggressive growth. In conclusion, our established new mouse model can contribute to increase knowledge about catastrophic PDAC. Citation Format: Takashi Yamaguchi, Sanae K. Ikehara, Hayao Nakanishi, Yuzuru Ikehara. Genetically engineered mouse models of catastrophic pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 822. doi:10.1158/1538-7445.AM2015-822

A Murine Myh6MerCreMer Knock-In Allele Specifically Mediates Temporal Genetic Deletion in Cardiomyocytes after Tamoxifen Induction.

A mouse model that mediates temporal, specific, and efficient myocardial deletion with Cre-LoxP technology will be a valuable tool to determine the function of genes during heart formation. Mhy6 encodes a cardiac muscle specific protein: alpha-myosin heavy chain. Here, we generated a new Myh6-MerCreMer (Myh6MerCreMer/+) inducible Cre knock-in mouse by inserting a MerCreMer cassette into the Myh6 start codon. By crossing knock-in mice with Rosa26 reporter lines, we found the Myh6MerCreMer/+ mice mediate complete Cre-LoxP recombination in cardiomyocytes after tamoxifen induction. X-gal staining and immunohistochemistry analysis revealed that Myh6-driven Cre recombinase was specifically activated in cardiomyocytes at embryonic and adult stages. Furthermore, echocardiography showed that Myh6MerCreMer/+ mice maintained normal cardiac structure and function before and after tamoxifen administration. These results suggest that the new Myh6MerCreMer/+ mouse can serve as a robust tool to dissect the roles of genes in heart development and function. Additionally, myocardial progeny during heart development and after cardiac injury can be traced using this mouse line.

Disruption of prostaglandin E2 receptor EP4 impairs urinary concentration via decreasing aquaporin 2 in renal collecting ducts

The antidiuretic hormone arginine vasopressin is a systemic effector in urinary concentration. However, increasing evidence suggests that other locally produced factors may also play an important role in the regulation of water reabsorption in renal collecting ducts. Recently, prostaglandin E2 (PGE2) receptor EP4 has emerged as a potential therapeutic target for the treatment of nephrogenic diabetes insipidus, but the underlying mechanism is unknown. To evaluate the role of EP4 in regulating water homeostasis, mice with renal tubule-specific knockout of EP4 (Ksp-EP4(-/-)) and collecting duct-specific knockout of EP4 (AQP2-EP4(-/-)) were generated using the Cre-loxP recombination system. Urine concentrating defect was observed in both Ksp-EP4(-/-) and AQP2-EP4(-/-) mice. Decreased aquaporin 2 (AQP2) abundance and apical membrane targeting in renal collecting ducts were evident in Ksp-EP4(-/-) mice. In vitro studies demonstrated that AQP2 mRNA and protein levels were significantly up-regulated in mouse primary inner medullary collecting duct (IMCD) cells after pharmacological activation or adenovirus-mediated overexpression of EP4 in a cAMP/cAMP-response element binding protein-dependent manner. In addition, EP4 activation or overexpression also increased AQP2 membrane accumulation in a mouse IMCD cell line (IMCD3) stably transfected with the AQP2 gene, mainly through the cAMP/protein kinase A and extracellular signal-regulated kinase pathways. In summary, the EP4 receptor in renal collecting ducts plays an important role in regulating urinary concentration under physiological conditions. The ability of EP4 to promote AQP2 membrane targeting and increase AQP2 abundance makes it a potential therapeutic target for the treatment of clinical disorders including acquired and congenital diabetes insipidus.

Optogenetic intervention to the vascular endothelium

Endothelium lining the interior of cardiovascular system and most visceral organs plays an important role in vascular function. Its dysfunction occurs in some of the most challenging diseases. An important function of the endothelium is to release vasoactive substances that act on the smooth muscle to change vascular tones. Substance secretion from endocrine cells relies on membrane potentials and firing activity, while it is unclear whether the membrane potential regulates substance release from the ECs. Understanding of this requires selective intervention to membrane potentials of the endothelial cells in situ. Here we show a novel intervention to endothelial cells using the optogenetic approach. A strain of transgenic mice was developed with the Cre–loxP recombination system. These transgenic mice expressed channelrhodopsin (ChR) in endothelial cells driven by the vascular endothelial cadherin or cdh5 promoter. Linked in a tandem with YFP, the ChR expression was detected by YFP fluorescence in various endothelium-lining tissues and organs. The YFP fluorescence was observed in the lumen of blood vessels and pericardium, but not in tissues beneath the endothelium lining. Optostimulation of dissociated endothelial cells evoked inward currents and depolarization. In the isolated and perfused heart, surprisingly, optostimulation of endothelial cells produced fast, robust, reproducible and long-lasting vasoconstriction that was not blocked by either ET-1A or TXA2 receptor antagonist. Similar optical vasoconstriction was found in the isolated and perfused kidney. These results indicate that the optogenetics is an effective intervention to vascular endothelium where optostimulation produces vasoconstriction.

Endothelin-1 critically influences cardiac function via superoxide-MMP9 cascade

We have generated low-expressing and high-expressing endothelin-1 genes (L and H) and have bred mice with four levels of expression: L/L, ∼20%; L/+, ∼65%; +/+ (wild type), 100%; and H/+, ∼350%. The hypomorphic L allele can be spatiotemporally switched to the hypermorphic H allele by Cre-loxP recombination. Young adult L/L and L/+ mice have dilated cardiomyopathy, hypertension, and increased plasma volumes, together with increased ventricular superoxide levels, increased matrix metalloproteinase 9 (Mmp9) expression, and reduced ventricular stiffness. H/+ mice have decreased plasma volumes and significantly heavy stiff hearts. Global or cardiomyocyte-specific switching expression from L to H normalized the abnormalities already present in young adult L/L mice. An epithelial sodium channel antagonist normalized plasma volume and blood pressure, but only partially corrected the cardiomyopathy. A superoxide dismutase mimetic made superoxide levels subnormal, reduced Mmp9 overexpression, and substantially improved cardiac function. Genetic absence of Mmp9 also improved cardiac function, but increased superoxide remained. We conclude that endothelin-1 is critical for maintaining normal contractile function, for controlling superoxide and Mmp9 levels, and for ensuring that the myocardium has sufficient collagen to prevent overstretching. Even a modest (∼35%) decrease in endothelin-1 gene (Edn1) expression is sufficient to cause cardiac dysfunction.