Severe acute respiratory syndrome (SARS) was first identified in Guangdong Province in China (28). Over the ensuing 9 months, more than 8,000 cases were identified throughout the world, with a ϳ10% case fatality rate. A novel coronavirus, SARS coronavirus (SARS-CoV), was identified as the causative agent (6,17,29,32). Initial investigations indicated that the virus spread to humans from infected exotic animals such as Himalayan palm civets (Paguma larvata) and Chinese ferret badgers (Melogale moschata) (12); more recent work has suggested that the natural reservoirs for the virus are wild bat populations in China (19,24). Although SARS has not recurred in human populations to a significant extent since 2003, the potential severity of such a recurrence has spurred interest in developing an animal model for the human disease.SARS-CoV infects and replicates in mice, ferrets, hamsters, and several species of nonhuman primates (cynomolgus and rhesus macaques, African green monkeys, and common marmosets) (reviewed in reference 37). However, none of these animals develop a clinical disease that is reproducible and equivalent in severity to that observed in SARS patients. A mouse model would be useful for answering many questions about SARS pathogenesis and for testing vaccine efficacy, in part because reagents for the study of the immune response are widely available. However, other than aged or immunocompromised (STAT1 Ϫ/Ϫ ) mice (37), these animals do not develop significant clinical disease, and lethality has not been demonstrated in any murine model of SARS. With the goal of developing a more robust murine model, we generated transgenic (Tg) mice in which expression of hACE2 (human angiotensin-converting enzyme 2, the primary host cell receptor for SARS-CoV [23]) was targeted to epithelial cells. While human ACE2 and murine ACE2 (mACE2) molecules are very homologous, mACE2 does not support SARS-CoV binding as efficiently as hACE2 (22). Here we show that the transgenic expression of hACE2 in epithelia converts a mild SARS-CoV infection into a rapidly fatal disease. MATERIALS AND METHODSMice. All animal studies were approved by the University of Iowa and the Veterans Administration Institutional Animal Care and Use committees. Mice transgenic for expression of hACE2 (K18-hACE2 mice) were generated as follows (see Fig. 1A). The hACE2 coding sequence was PCR amplified from IMAGE consortium clone ID 5243048 (ATCC, Manassas, VA) and cloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA). The lacZ coding sequence in the previously described pK18mTElacZ-K18i6x7pA construct (16) (a kind gift from Jim Hu, Hospital for Sick Children, Toronto, Canada) was then replaced by the hACE2 coding sequence to create pK18-hACE2. 5Ј of the hACE2 coding sequence, this plasmid contains 2.5 kb of upstream genomic sequence, the promoter, and the first intron (with a mutation in the 3Ј splice acceptor site to reduce exon skipping) of the human cytokeratin 18 (K18) gene as well as a translational enhancer sequence from alfalfa mosaic vi...

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor that plays a critical role in metabolism. Thiazolidinediones, high-affinity PPARgamma ligands used clinically to treat type II diabetes, have been reported to lower blood pressure and provide other cardiovascular benefits. Some mutations in PPARgamma (PPARG) cause type II diabetes and severe hypertension. Here we tested the hypothesis that PPARgamma in vascular muscle plays a role in the regulation of vascular tone and blood pressure. Transgenic mice expressing dominant-negative mutations in PPARgamma under the control of a smooth-muscle-specific promoter exhibit a loss of responsiveness to nitric oxide and striking alterations in contractility in the aorta, hypertrophy and inward remodeling in the cerebral microcirculation, and systolic hypertension. These results identify PPARgamma as pivotal in vascular muscle as a regulator of vascular structure, vascular function, and blood pressure, potentially explaining some of the cardioprotective effects of thiazolidinediones.

T he renin-angiotensin system (RAS) is well known for its physiological and pathophysiological roles in the regulation of blood pressure (BP) and cardiovascular function. 1,2 A new component of the RAS, angiotensin-converting enzyme (ACE) type 2 has been identified, from human heart failure ventricle and lymphoma cDNA libraries (reviewed elsewhere 3,4 ). Although the angiotensin-converting enzyme type 2 (ACE2) transcript was first described in heart, kidney and testis, additional studies reported ACE2 mRNA in rat medulla oblongata 4 and ACE2 activity in mouse brain. 5 Recently, we showed the presence of both ACE2 protein and mRNA widespread throughout the murine brain, in regions involved in the central regulation of cardiovascular function as well as noncardiovascular regions. 6 ACE2 converts Ang II into the vasodilatory peptide Ang-(1-7) with an affinity 400-fold higher than for Ang I. 7 In the central nervous system (CNS), Ang-(1-7) has been shown to enhance sensitivity of the bradycardic component of the cardiac baroreceptor reflex 8 and to promote vasodilation in hypertensive animals. 9,10 As a key enzyme in generating Ang-(1-7), ACE2 is thought to be a pivotal player in central BP regulation. 3,5 Several evidences from various laboratories have shown the beneficial effects of peripheral ACE2 in the regulation of cardiovascular hypertrophy and BP control. 10 -12 In the CNS, using a lentivirus coding for ACE2, Yamazato et al previously showed that ACE2 overexpression in the rostral ventrolateral medulla, could reverse hypertension in spontaneously hypertensive rats (SHR). 13 More recently, we reported that brain-targeted ACE2 overexpression in the subfornical organ (SFO) prevents the acute Ang II-mediated pressor and Original

Peripheral nerve damage initiates a complex series of structural and cellular processes that culminate in chronic neuropathic pain. The recent success of a type 2 angiotensin II (Ang II) receptor (AT2R) antagonist in a phase II clinical trial for the treatment of postherpetic neuralgia suggests angiotensin signaling is involved in neuropathic pain. However, transcriptome analysis indicates a lack of AT2R gene () expression in human and rodent sensory ganglia, raising questions regarding the tissue/cell target underlying the analgesic effect of AT2R antagonism. We show that selective antagonism of AT2R attenuates neuropathic but not inflammatory mechanical and cold pain hypersensitivity behaviors in mice. -expressing macrophages (MΦs) constitute the predominant immune cells that infiltrate the site of nerve injury. Interestingly, neuropathic mechanical and cold pain hypersensitivity can be attenuated by chemogenetic depletion of peripheral MΦs and AT2R-null hematopoietic cell transplantation. Our study identifies AT2R on peripheral MΦs as a critical trigger for pain sensitization at the site of nerve injury, and therefore proposes a translatable peripheral mechanism underlying chronic neuropathic pain.

Thoracic aortic aneurysms and dissections (TAAD) represent a substantial cause of morbidity and mortality worldwide. Many individuals presenting with an inherited form of TAAD do not have causal mutations in the set of genes known to underlie disease. Using whole-genome sequencing in two first cousins with TAAD, we identified a missense mutation in the lysyl oxidase (LOX) gene (c.893T > G encoding p.Met298Arg) that cosegregated with disease in the family. Using clustered regularly interspaced short palindromic repeats (CRISPR)/clustered regularly interspaced short palindromic repeats-associated protein-9 nuclease (Cas9) genome engineering tools, we introduced the human mutation into the homologous position in the mouse genome, creating mice that were heterozygous and homozygous for the human allele. Mutant mice that were heterozygous for the human allele displayed disorganized ultrastructural properties of the aortic wall characterized by fragmented elastic lamellae, whereas mice homozygous for the human allele died shortly after parturition from ascending aortic aneurysm and spontaneous hemorrhage. These data suggest that a missense mutation in LOX is associated with aortic disease in humans, likely through insufficient cross-linking of elastin and collagen in the aortic wall. Mutation carriers may be predisposed to vascular diseases because of weakened vessel walls under stress conditions. LOX sequencing for clinical TAAD may identify additional mutation carriers in the future. Additional studies using our mouse model of LOX-associated TAAD have the potential to clarify the mechanism of disease and identify novel therapeutics specific to this genetic cause.

Abstract-The transcription factor PPAR␥ is expressed in endothelium and vascular muscle where it may exert antiinflammatory and antioxidant effects. We tested the hypothesis that PPAR␥ plays a protective role in the vasculature by examining vascular structure and function in heterozygous knockin mice expressing the P465L dominant negative mutation in PPAR␥ (L/ϩ). In L/ϩ aorta, responses to the endothelium-dependent agonist acetylcholine (ACh) were not affected, but there was an increase in contraction to serotonin, PGF 2␣ , and endothelin-1. In cerebral blood vessels both in vitro and in vivo, ACh produced dilation that was markedly impaired in L/ϩ mice. Superoxide levels were elevated in cerebral arterioles from L/ϩ mice and responses to ACh were restored to normal with a scavenger of superoxide. Diameter of maximally dilated cerebral arterioles was less, whereas wall thickness and cross-sectional area was greater in L/ϩ mice, indicating cerebral arterioles underwent hypertrophy and remodeling. Thus, interference with PPAR␥ signaling produces endothelial dysfunction via a mechanism involving oxidative stress and causes vascular hypertrophy and inward remodeling. These findings indicate that PPAR␥ has vascular effects which are particularly profound in the cerebral circulation and provide genetic evidence that PPAR␥ plays a critical role in protecting blood vessels. (Hypertension. 2008;51:867-871.)Key Words: endothelial function Ⅲ dominant negative Ⅲ hypertension Ⅲ remodeling Ⅲ hypertrophy T he peroxisome proliferator-activated receptor gamma (PPAR␥) is a ligand-activated transcription factor which has gained prominence because of its involvement in complex diseases such as diabetes, obesity, atherosclerosis, and hypertension. Recent interest in the role of PPAR␥ in the vasculature has substantially increased because of the antiinflammatory and antioxidant effects reported for PPAR␥ agonists (reviewed by Schiffrin et al 1 ). Naturally occurring mutations in humans, resulting in either constitutive activation or impairment of PPAR␥ function, strongly support its physiological importance and illustrate the severe consequences for cardiovascular related events when PPAR␥ signaling is altered. 2 Individuals with dominant negative mutations in PPAR␥ present with early onset hypertension and elements of the metabolic syndrome. 2 Although the importance of PPAR␥ in adipose tissue is now well documented, its role in the cardiovascular system has only begun to emerge. PPAR␥ is the molecular target of the thiazolidinediones (TZDs) class of antidiabetes drugs. These drugs increase insulin sensitivity but also lower blood pressure in patients with type 2 diabetes 3 and in animal models of hypertension. 4 TZDs also improve endothelial function and reduce blood pressure in nondiabetic models of hypertension, underscoring the potential protective effects of PPAR␥ in the vessel wall. 5 PPAR␥ is expressed in endothelium and vascular muscle and there is growing evidence that PPAR␥ may have direct effects in the vasculature. 6 ...

Abstract-The ligand-activated transcription factor peroxisome proliferator activated receptor gamma (PPAR␥) is expressed in vascular endothelium where it exerts anti-inflammatory and antioxidant effects. However, its role in regulating vascular function remains undefined. We examined endothelial function in transgenic mice expressing dominant-negative mutants of PPAR␥ under the control of an endothelial-specific promoter to test the hypothesis that endothelial PPAR␥ plays a protective role in the vasculature. Under baseline conditions, responses to the endotheliumdependent agonist acetylcholine were not affected in either aorta or the basilar artery in vitro. In response to feeding a high-fat diet for 12 weeks, acetylcholine produced dilation that was markedly impaired in the basilar artery of mice expressing dominant-negative mutants, but not in mice expressing wild-type PPAR␥ controlled by the same promoter. Unlike basilar artery, 12 weeks of a high-fat diet was not sufficient to cause endothelial dysfunction in the aorta of mice expressing dominant-negative PPAR␥, although aortic dysfunction became evident after 25 weeks. The responses to acetylcholine in basilar artery were restored to normal after treatment with a scavenger of superoxide. Baseline blood pressure was only slightly elevated in the transgenic mice, but the pressor response to angiotensin II was augmented. Thus, interference with PPAR␥ in the endothelium produces endothelial dysfunction in the cerebral circulation through a mechanism involving oxidative stress. Consistent with its role as a fatty acid sensor, these findings provide genetic evidence that endothelial PPAR␥ plays a critical role in protecting blood vessels in response to a high-fat diet. Key Words: endothelium Ⅲ oxidative stress Ⅲ transcription Ⅲ transgenic animals Ⅲ vascular P eroxisome proliferator activated receptor gamma (PPAR␥) is a ligand-activated transcription factor targeted by the thiazolidinedione (TZD) class of antidiabetes medications. Activation of PPAR␥ by TZDs improves insulin sensitivity and lowers blood pressure in type II diabetes, whereas individuals with dominant-negative mutations in PPAR␥ present with severe insulin resistance, type II diabetes, and early-onset hypertension. 1 Studies in humans and animals suggest that TZDs are generally cardioprotective, although their clinical safety has been recently challenged. 2 That TZDs can lower blood pressure in the face of weight gain and water and salt retention by the kidneys suggests that the antihypertensive effects may be particularly profound.Heterozygous mice carrying one normal PPAR␥ allele and one dominant-negative allele (L/ϩ mice) exhibit a moderate increase in blood pressure. 3,4 We have recently reported that L/ϩ mice exhibit endothelial dysfunction and hypertrophy and inward remodeling in the cerebral vasculature. 4 Oxidative stress was the basis of endothelial dysfunction in the model as superoxide was increased and vascular function was restored to normal by a free radical scavenger. These data prov...

Cantu syndrome (CS) is characterized by multiple vascular and cardiac abnormalities including vascular dilation and tortuosity, systemic hypotension, and cardiomegaly. The disorder is caused by gain-of-function (GOF) mutations in genes encoding pore-forming (Kir6.1, KCNJ8) and accessory (SUR2, ABCC9) ATP-sensitive potassium (KATP) channel subunits. However, there is little understanding of the link between molecular dysfunction and the complex pathophysiology observed, and there is no known treatment, in large part due to the lack of appropriate preclinical disease models in which to test therapies. Notably, expression of Kir6.1 and SUR2 does not fully overlap, and the relative contribution of KATP GOF in various cardiovascular tissues remains to be elucidated. To investigate pathophysiologic mechanisms in CS we have used CRISPR/Cas9 engineering to introduce CS-associated SUR2[A478V] and Kir6.1[V65M] mutations to the equivalent endogenous loci in mice. Mirroring human CS, both of these animals exhibit low systemic blood pressure and dilated, compliant blood vessels, as well dramatic cardiac enlargement, the effects being more severe in V65M animals than in A478V animals. In both animals, whole-cell patch-clamp recordings reveal enhanced basal KATP conductance in vascular smooth muscle, explaining vasodilation and lower blood pressure, and demonstrating a cardinal role for smooth muscle KATP dysfunction in CS etiology. Echocardiography confirms in situ cardiac enlargement and increased cardiac output in both animals. Patch-clamp recordings reveal reduced ATP sensitivity of ventricular myocyte KATP channels in A478V, but normal ATP sensitivity in V65M, suggesting that cardiac remodeling occurs secondary to KATP overactivity outside of the heart. These SUR2[A478V] and Kir6.1[V65M] animals thus reiterate the key cardiovascular features seen in human CS. They establish the molecular basis of the pathophysiological consequences of reduced smooth muscle excitability resulting from SUR2/Kir6.1-dependent KATP GOF, and provide a validated animal model in which to examine potential therapeutic approaches to treating CS.