Mark C. Fishman

Targeted disruption of the neuronal nitric oxide synthase gene

By homologous recombination, we have generated mice that lack the neuronal nitric oxide synthase (NOS) gene. Neuronal NOS expression and NADPH-diaphorase (NDP) staining are absent in the mutant mice. Very low level residual catalytic activity suggests that other enzymes in the brain may generate nitric oxide. The neurons normally expressing NOS appear intact, and the mutant NOS mice are viable, fertile, and without evident histopathological abnormalities in the central nervous system. The most evident effect of disrupting the neuronal NOS gene is the development of grossly enlarged stomachs, with hypertrophy of the pyloric sphincter and the circular muscle layer. This phenotype resembles the human disorder infantile pyloric stenosis, in which gastric outlet obstruction is associated with the lack of NDP neurons in the pylorus.

Nitric oxide synthase modulates angiogenesis in response to tissue ischemia.

We tested the hypothesis that endothelial nitric oxide synthase (eNOS) modulates angiogenesis in two animal models in which therapeutic angiogenesis has been characterized as a compensatory response to tissue ischemia. We first administered L-arginine, previously shown to augment endogenous production of NO, to normal rabbits with operatively induced hindlimb ischemia. Angiogenesis in the ischemic hindlimb was significantly improved by dietary supplementation with L-arginine, compared to placebo-treated controls; angiographically evident vascularity in the ischemic limb, hemodynamic indices of limb perfusion, capillary density, and vasomotor reactivity in the collateral vessel-dependent ischemic limb were all improved by oral L-arginine supplementation. A murine model of operatively induced hindlimb ischemia was used to investigate the impact of targeted disruption of the gene encoding for ENOS on angiogenesis. Angiogenesis in the ischemic hindlimb was significantly impaired in eNOS-/- mice versus wild-type controls evaluated by either laser Doppler flow analysis or capillary density measurement. Impaired angiogenesis in eNOS-/- mice was not improved by administration of vascular endothelial growth factor (VEGF), suggesting that eNOS acts downstream from VEGF. Thus, (a) eNOS is a downstream mediator for in vivo angiogenesis, and (b) promoting eNOS activity by L-arginine supplementation accelerates in vivo angiogenesis. These findings suggest that defective endothelial NO synthesis may limit angiogenesis in patients with endothelial dysfunction related to atherosclerosis, and that oral L-arginine supplementation constitutes a potential therapeutic strategy for accelerating angiogenesis in patients with advanced vascular obstruction.

Enlarged Infarcts in Endothelial Nitric Oxide Synthase Knockout Mice are Attenuated by Nitro-L-Arginine:

Infarct size and vascular hemodynamics were measured 24 h after middle cerebral artery (MCA) occlusion in mice genetically deficient in the endothelial nitric oxide synthase (eNOS) isoform. eNOS mutant mice developed larger infarcts (21%) than the wild-type strain when assessed 24 h after intraluminal filament occlusion. Moreover, regional CBF values recorded in the MCA territory by laser-Doppler flowmetry were more severely reduced after occlusion and were disproportionately reduced during controlled hemorrhagic hypotension in autoregulation experiments. Unlike the situation in wild-type mice, nitro-L-arginine superfusion (1 mM) dilated pial arterioles of eNOS knockout mice in a closed cranial window preparation. As noted previously, eNOS mutant mice were hypertensive. However, infarct size remained increased despite lowering blood pressure to normotensive levels by hydralazine treatment. Systemic administration of nitro-L-arginine decreased infarct size in eNOS mutant mice (24%) but not in the wild-type strain. This finding complements published data showing that nitro-L-arginine increases infarct size in knockout mice expressing the eNOS but not the neuronal NOS isoform (i.e., neuronal NOS knockout mice). We conclude that NO production within endothelium may protect brain tissue, perhaps by hemodynamic mechanisms, whereas neuronal NO overproduction may lead to neurotoxicity.

Impaired Defense of Intestinal Mucosa in Mice Lacking Intestinal Trefoil Factor

The mechanisms that maintain the epithelial integrity of the gastrointestinal tract remain largely undefined. The gene encoding intestinal trefoil factor (ITF), a protein secreted throughout the small intestine and colon, was rendered nonfunctional in mice by targeted disruption. Mice lacking ITF had impaired mucosal healing and died from extensive colitis after oral administration of dextran sulfate sodium, an agent that causes mild epithelial injury in wild-type mice. ITF-deficient mice manifested poor epithelial regeneration after injury. These findings reveal a central role for ITF in the maintenance and repair of the intestinal mucosa.

Gridlock signalling pathway fashions the first embryonic artery

Arteries and veins are morphologically, functionally and molecularly very different, but how this distinction is established during vasculogenesis is unknown. Here we show, by lineage tracking in zebrafish embryos, that angioblast precursors for the trunk artery and vein are spatially mixed in the lateral posterior mesoderm. Progeny of each angioblast, however, are restricted to one of the vessels. This arterial–venous decision is guided by gridlock (grl), an artery-restricted gene that is expressed in the lateral posterior mesoderm. Graded reduction of grl expression, by mutation or morpholino antisense, progressively ablates regions of the artery, and expands contiguous regions of the vein, preceded by an increase in expression of the venous marker EphB4 receptor (ephb4) and diminution of expression of the arterial marker ephrin-B2 (efnb2). grl is downstream of notch, and interference with notch signalling, by blocking Su(H), similarly reduces the artery and increases the vein. Thus, a notch–grl pathway controls assembly of the first embryonic artery, apparently by adjudicating an arterial versus venous cell fate decision.

Cardiac troponin T is essential in sarcomere assembly and cardiac contractility.

Mutations of the gene (TNNT2) encoding the thin-filament contractile protein cardiac troponin T are responsible for 15% of all cases of familial hypertrophic cardiomyopathy, the leading cause of sudden death in young athletes. Mutant proteins are thought to act through a dominant-negative mode that impairs function of heart muscle. TNNT2 mutations can also lead to dilated cardiomyopathy, a leading cause of heart failure. Despite the importance of cardiac troponin T in human disease, its loss-of-function phenotype has not been described. We show that the zebrafish silent heart (sih) mutation affects the gene tnnt2. We characterize two mutated alleles of sih that severely reduce tnnt2 expression: one affects mRNA splicing, and the other affects gene transcription. Tnnt2, together with α-tropomyosin (Tpma) and cardiac troponins C and I (Tnni3), forms a calcium-sensitive regulatory complex within sarcomeres. Unexpectedly, in addition to loss of Tnnt2 expression in sih mutant hearts, we observed a significant reduction in Tpma and Tnni3, and consequently, severe sarcomere defects. This interdependence of thin-filament protein expression led us to postulate that some mutations in tnnt2 may trigger misregulation of thin-filament protein expression, resulting in sarcomere loss and myocyte disarray, the life-threatening hallmarks of TNNT2 mutations in mice and humans.

Long-term potentiation is reduced in mice that are doubly mutant in endothelial and neuronal nitric oxide synthase.

Nitric oxide (NO) has been implicated in hippocampal long-term potentiation (LTP), but LTP is normal in mice with a targeted mutation in the neuronal form of NO synthase (nNOS-). LTP was also normal in mice with a targeted mutation in endothelial NOS (eNOS-), but LTP in stratum radiatum of CA1 was significantly reduced in doubly mutant mice (nNOS-/eNOS-). By contrast, LTP in stratum oriens was normal in the doubly mutant mice. These results provide the first genetic evidence that NOS is involved in LTP in stratum radiatum and suggest that the neuronal and endothelial forms can compensate for each other in mice with a single mutation. They further suggest that there is also a NOS-independent component of LTP in stratum radiatum and that LTP in stratum oriens is largely NOS independent.

Reduced Brain Edema and Infarction Volume in Mice Lacking the Neuronal Isoform of Nitric Oxide Synthase after Transient MCA Occlusion

Infarct volume and edema were assessed after transient focal ischemia in mice lacking neuronal nitric oxide synthase (NOS) gene expression. With use of an 8–0 coated monofilament, the middle cerebral artery (MCA) of mutant (n = 32) and wild-type mice [SV-129 (n = 31), C57Black/6 (n = 18)] were occluded for 3 h and reperfused for up to 24 h. Regional CBF (rCBF), neurological deficits, water content, and infarct volume were examined in all three strains. rCBF, blood pressure, and heart rate did not differ between groups when measured for 1 h after reperfusion. Neurological deficits were less severe in mutant mice after MCA occlusion. Brain water content at 3 h after reperfusion and infarct volume at 24 h after reperfusion were greater in wild-type mice. These data indicate that genetic deletion of neuronal NOS confers resistance to focal ischemic injury in a reperfusion model. The findings agree with previous studies showing that tissue injury is less extensive after both permanent MCA occlusion and global ischemia in mice lacking neuronal NOS gene expression. Hence, NO may play a pivotal role in the pathogenesis of ischemic brain damage.

Interaction of genetic deficiency of endothelial nitric oxide, gender, and pregnancy in vascular response to injury in mice.

To begin to dissect atherogenesis as a complex genetic disorder affected by genetic makeup and environment, we have (a) generated a reproducible mouse model of neointimal growth; (b) evaluated the effect of disruption of a single gene, endothelial nitric oxide synthase, believed to be central to intimal growth, and (c) examined the modifying effects of gender and pregnancy upon the vascular response. Cuff placement around the femoral artery causes reproducible intimal growth. We assessed the response to injury by quantitative morphometry, measuring the intimal to medial (I/M) volume ratio. In wild-type mice, cuff placement causes pronounced intimal proliferation without affecting the media, resulting in I/M ratios of 31% (SV129 males) and 27% (C57BL/6 males). eNOS mutant male mice have a much greater degree of intimal growth (I/M ratio of 70%). Female mice show less intimal response than do males, although eNOS mutant female mice still have more response than do wild-type females. Most dramatic, however, is the effect of pregnancy, which essentially abolishes the intimal response to injury, even overriding the effect of eNOS mutation. We conclude that eNOS deficiency is a genetic predisposition to intimal proliferation that is enhanced by male gender, and that may be overridden by pregnancy.

Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation

Conventional drug discovery approaches require a priori selection of an appropriate molecular target, but it is often not obvious which biological pathways must be targeted to reverse a disease phenotype. Phenotype-based screens offer the potential to identify pathways and potential therapies that influence disease processes. The zebrafish mutation gridlock (grl, affecting the gene hey2) disrupts aortic blood flow in a region and physiological manner akin to aortic coarctation in humans. Here we use a whole-organism, phenotype-based, small-molecule screen to discover a class of compounds that suppress the coarctation phenotype and permit survival to adulthood. These compounds function during the specification and migration of angioblasts. They act to upregulate expression of vascular endothelial growth factor (VEGF), and the activation of the VEGF pathway is sufficient to suppress the gridlock phenotype. Thus, organism-based screens allow the discovery of small molecules that ameliorate complex dysmorphic syndromes even without targeting the affected gene directly.