Showing papers in "Journal of Clinical Investigation in 2000"

Obesity and insulin resistance

Abstract: The association of obesity with type 2 diabetes has been recognized for decades, and the major basis for this link is the ability of obesity to engender insulin resistance. Insulin resistance is a fundamental aspect of the etiology of type 2 diabetes and is also linked to a wide array of other pathophysiologic sequelae including hypertension, hyperlipidemia, atherosclerosis (i.e., the metabolic syndrome, or syndrome X), and polycystic ovarian disease (1). Although many details of the mechanisms by which the enlarged adipose tissue mass that defines obesity causes systemic insulin resistance remain unknown, the past several years have witnessed an explosive increase in our understanding of what may now be referred to as the adipo-insulin axis. There are also grounds for considering the related possibility that insulin resistance and hyperinsulinemia, in addition to being caused by obesity, can contribute to the development of obesity. In this Perspective, we will review recent progress, highlight areas of controversy or uncertainty, and suggest approaches to clarifying the unresolved issues.

Cellular mechanisms of insulin resistance

Abstract: It is estimated that by the year 2020 there will be approximately 250 million people affected by type 2 diabetes mellitus worldwide (1). Although the primary factors causing this disease are unknown, it is clear that insulin resistance plays a major role in its development. Evidence for this comes from (a) the presence of insulin resistance 10–20 years before the onset of the disease (2, 3); (b) cross-sectional studies demonstrating that insulin resistance is a consistent finding in patients with type 2 diabetes (3–6); and (c) prospective studies demonstrating that insulin resistance is the best predictor of whether or not an individual will later become diabetic (2, 3). Here, I focus on some recent advances in our understanding of human insulin resistance that have been made using nuclear magnetic resonance spectroscopy (NMR). This technique takes advantage of the spin properties of the nuclei of certain isotopes, such as 1H, 13C, and 31P, which endow the isotopes with a magnetic component that can be used to measure the concentration of intracellular metabolites noninvasively and to assess biochemical differences between normal and diabetic subjects. Drawing on NMR studies from my laboratory and others, I first consider the control of glucose phosphorylation and transport in regulating muscle responses to insulin. I then turn to the effects of fatty acids on insulin responses, showing that commonly accepted models that attempt to explain the association of insulin resistance and obesity are incompatible with recent findings. Finally, I propose an alternative model that appears to fit these and other available data.

Origins of circulating endothelial cells and endothelial outgrowth from blood

Abstract: Normal adults have a small number of circulating endothelial cells (CEC) in peripheral blood, and endothelial outgrowth has been observed from cultures of blood. In this study we seek insight into the origins of CEC and endothelial outgrowth from cultures of blood. Fluorescence in situ hybridization analysis of blood samples from bone marrow transplant recipients who had received gender-mismatched transplants 5-20 months earlier showed that most CEC in fresh blood had recipient genotype. Endothelial outgrowth from the same blood samples after 9 days in culture (5-fold expansion) was still predominantly of the recipient genotype. In contrast, endothelial outgrowth after approximately 1 month (102-fold expansion) was mostly of donor genotype. Thus, recipient-genotype endothelial cells expanded only approximately 20-fold over this period, whereas donor-genotype endothelial cells expanded approximately 1000-fold. These data suggest that most CEC in fresh blood originate from vessel walls and have limited growth capability. Conversely, the data indicate that outgrowth of endothelial cells from cultures of blood is mostly derived from transplantable marrow-derived cells. Because these cells have more delayed outgrowth but a greater proliferative rate, our data suggest that they are derived from circulating angioblasts.

Clearance of Alzheimer's amyloid-ss(1-40) peptide from brain by LDL receptor-related protein-1 at the blood-brain barrier.

Abstract: Elimination of amyloid-ss peptide (Ass) from the brain is poorly understood. After intracerebral microinjections in young mice, (125)I-Ass(1-40) was rapidly removed from the brain (t(1/2)

TNF-α induces osteoclastogenesis by direct stimulation of macrophages exposed to permissive levels of RANK ligand

Abstract: While TNF-α is pivotal to the pathogenesis of inflammatory osteolysis, the means by which it recruits osteoclasts and promotes bone destruction are unknown. We find that a pure population of murine osteoclast precursors fails to undergo osteoclastogenesis when treated with TNF-α alone. In contrast, the cytokine dramatically stimulates differentiation in macrophages primed by less than one percent of the amount of RANKL (ligand for the receptor activator of NF-κB) required to induce osteoclast formation. Mirroring their synergistic effects on osteoclast differentiation, TNF-α and RANKL markedly potentiate NF-κB and stress-activated protein kinase/c-Jun NH2-terminal kinase activity, two signaling pathways essential for osteoclastogenesis. In vivo administration of TNF-α prompts robust osteoclast formation in chimeric animals in which β-galactosidase positive, TNF-responsive macrophages develop within a TNF-nonresponsive stromal environment. Thus, while TNF-α alone does not induce osteoclastogenesis, it does so both in vitro and in vivo by directly targeting macrophages within a stromal environment that expresses permissive levels of RANKL. Given the minuscule amount of RANKL sufficient to synergize with TNF-α to promote osteoclastogenesis, TNF-α appears to be a more convenient target in arresting inflammatory osteolysis.

Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E–deficient mice

Abstract: Increased plasma concentrations of angiotension II (Ang II) have been implicated in atherogenesis. To examine this relationship directly, we infused Ang II or vehicle for 1 month via osmotic minipumps into mature apoE(-/-) mice. These doses of Ang II did not alter arterial blood pressure, body weight, serum cholesterol concentrations, or distribution of lipoprotein cholesterol. However, Ang II infusions promoted an increased severity of aortic atherosclerotic lesions. These Ang II-induced lesions were predominantly lipid-laden macrophages and lymphocytes; moreover, Ang II promoted a marked increase in the number of macrophages present in the adventitial tissue underlying lesions. Unexpectedly, pronounced abdominal aortic aneurysms were present in apoE(-/-) mice infused with Ang II. Sequential sectioning of aneurysmal abdominal aorta revealed two major characteristics: an intact artery that is surrounded by a large remodeled adventitia, and a medial break with pronounced dilation and more modestly remodeled adventitial tissue. Although no atherosclerotic lesions were visible at the medial break point, the presence of hyperlipidemia was required because infusions of Ang II into apoE(+/+) mice failed to generate aneurysms. These results demonstrate that increased plasma concentrations of Ang II have profound and rapid effects on vascular pathology when combined with hyperlipidemia, in the absence of hemodynamic influences.

Peroxisome proliferator–activated receptor γ coactivator-1 promotes cardiac mitochondrial biogenesis

Abstract: Cardiac mitochondrial function is altered in a variety of inherited and acquired cardiovascular diseases. Recent studies have identified the transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1) as a regulator of mitochondrial function in tissues specialized for thermogenesis, such as brown adipose. We sought to determine whether PGC-1 controlled mitochondrial biogenesis and energy-producing capacity in the heart, a tissue specialized for high-capacity ATP production. We found that PGC-1 gene expression is induced in the mouse heart after birth and in response to short-term fasting, conditions known to increase cardiac mitochondrial energy production. Forced expression of PGC-1 in cardiac myocytes in culture induced the expression of nuclear and mitochondrial genes involved in multiple mitochondrial energy-transduction/energy-production pathways, increased cellular mitochondrial number, and stimulated coupled respiration. Cardiac-specific overexpression of PGC-1 in transgenic mice resulted in uncontrolled mitochondrial proliferation in cardiac myocytes leading to loss of sarcomeric structure and a dilated cardiomyopathy. These results identify PGC-1 as a critical regulatory molecule in the control of cardiac mitochondrial number and function in response to energy demands.

VEGF enhances angiogenesis and promotes blood-brain barrier leakage in the ischemic brain

Abstract: VEGF is a secreted mitogen associated with angiogenesis and is also a potent vascular permeability factor. The biological role of VEGF in the ischemic brain remains unknown. This study was undertaken to investigate whether VEGF enhances cerebral microvascular perfusion and increases blood-brain barrier (BBB) leakage in the ischemic brain. Using magnetic resonance imaging (MRI), three-dimensional laser-scanning confocal microscope, and functional neurological tests, we measured the effects of administrating recombinant human VEGF(165) (rhVEGF(165)) on angiogenesis, functional neurological outcome, and BBB leakage in a rat model of focal cerebral embolic ischemia. Late (48 hours) administration of rhVEGF(165) to the ischemic rats enhanced angiogenesis in the ischemic penumbra and significantly improved neurological recovery. However, early postischemic (1 hour) administration of rhVEGF(165) to ischemic rats significantly increased BBB leakage, hemorrhagic transformation, and ischemic lesions. Administration of rhVEGF(165) to ischemic rats did not change BBB leakage and cerebral plasma perfusion in the contralateral hemisphere. Our results indicate that VEGF can markedly enhance angiogenesis in the ischemic brain and reduce neurological deficits during stroke recovery and that inhibition of VEGF at the acute stage of stroke may reduce the BBB permeability and the risk of hemorrhagic transformation after focal cerebral ischemia.

Insulin resistance and cardiovascular disease

Abstract: Clearly, insulin resistance is not simply a problem of deficient glucose uptake in response to insulin, but a multifaceted syndrome that increases significantly the risk for cardiovascular disease. The links between insulin resistance and the associated dyslipidemia, hypertension, hypercoagulability, and atherosclerosis are numerous and complex. This complexity derives both from the almost certain multiple causes of the insulin resistance syndrome and from the interaction of genes predisposing to insulin resistance with other genes that have their own, independent impact on lipid metabolism, blood pressure regulation, coagulation, and artery wall biology. Nonetheless, I suggest that dysregulation of fatty acid metabolism plays a central role in the development of this phenotype. Thus, the association between insulin resistance and dyslipidemia is clearly initiated by increased FFA release from, or defective uptake of FFAs into, adipocytes. Recent studies linking fatty acids to endothelial dysfunction, together with the clear role of VLDL in the stimulation of PAI-1, further support the view that dysregulation of fatty acid metabolism sits close to the center of the pathophysiology of the insulin resistance syndrome, at least as it relates to risk for cardiovascular disease.

Edg-1, the G protein–coupled receptor for sphingosine-1-phosphate, is essential for vascular maturation

Abstract: Sphingolipid signaling pathways have been implicated in many critical cellular events. Sphingosine-1-phosphate (SPP), a sphingolipid metabolite found in high concentrations in platelets and blood, stimulates members of the endothelial differentiation gene (Edg) family of G protein-coupled receptors and triggers diverse effects, including cell growth, survival, migration, and morphogenesis. To determine the in vivo functions of the SPP/Edg signaling pathway, we disrupted the Edg1 gene in mice. Edg1(-/-) mice exhibited embryonic hemorrhage leading to intrauterine death between E12.5 and E14.5. Vasculogenesis and angiogenesis appeared normal in the mutant embryos. However, vascular maturation was incomplete due to a deficiency of vascular smooth muscle cells/pericytes. We also show that Edg-1 mediates an SPP-induced migration response that is defective in mutant cells due to an inability to activate the small GTPase, Rac. Our data reveal Edg-1 to be the first G protein-coupled receptor required for blood vessel formation and show that sphingolipid signaling is essential during mammalian development.

Inhibition of VEGF receptors causes lung cell apoptosis and emphysema

Abstract: Pulmonary emphysema, a significant global health problem, is characterized by a loss of alveolar structures. Because VEGF is a trophic factor required for the survival of endothelial cells and is abundantly expressed in the lung, we hypothesized that chronic blockade of VEGF receptors could induce alveolar cell apoptosis and emphysema. Chronic treatment of rats with the VEGF receptor blocker SU5416 led to enlargement of the air spaces, indicative of emphysema. The VEGF receptor inhibitor SU5416 induced alveolar septal cell apoptosis but did not inhibit lung cell proliferation. Viewed by angiography, SU5416-treated rat lungs showed a pruning of the pulmonary arterial tree, although we observed no lung infiltration by inflammatory cells or fibrosis. SU5416 treatment led to a decrease in lung expression of VEGF receptor 2 (VEGFR-2), phosphorylated VEGFR-2, and Akt-1 in the complex with VEGFR-2. Treatment with the caspase inhibitor Z-Asp-CH2-DCB prevented SU5416-induced septal cell apoptosis and emphysema development. These findings suggest that VEGF receptor signaling is required for maintenance of the alveolar structures and, further, that alveolar septal cell apoptosis contributes to the pathogenesis of emphysema.

Insulin resistance differentially affects the PI 3-kinase- and MAP kinase-mediated signaling in human muscle.

Abstract: The broad nature of insulin resistant glucose metabolism in skeletal muscle of patients with type 2 diabetes suggests a defect in the proximal part of the insulin signaling network. We sought to identify the pathways compromised in insulin resistance and to test the effect of moderate exercise on whole-body and cellular insulin action. We conducted euglycemic clamps and muscle biopsies on type 2 diabetic patients, obese nondiabetics and lean controls, with and without a single bout of exercise. Insulin stimulation of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway, as measured by phosphorylation of the insulin receptor and IRS-1 and by IRS protein association with p85 and with PI 3-kinase, was dramatically reduced in obese nondiabetics and virtually absent in type 2 diabetic patients. Insulin stimulation of the MAP kinase pathway was normal in obese and diabetic subjects. Insulin stimulation of glucose-disposal correlated with association of p85 with IRS-1. Exercise 24 hours before the euglycemic clamp increased phosphorylation of insulin receptor and IRS-1 in obese and diabetic subjects but did not increase glucose uptake or PI 3-kinase association with IRS-1 upon insulin stimulation. Thus, insulin resistance differentially affects the PI 3-kinase and MAP kinase signaling pathways, and insulin-stimulated IRS-1-association with PI 3-kinase defines a key step in insulin resistance.

Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity

Abstract: Various conventional chemotherapeutic drugs can block angiogenesis or even kill activated, dividing endothelial cells. Such effects may contribute to the antitumor efficacy of chemotherapy in vivo and may delay or prevent the acquisition of drug-resistance by cancer cells. We have implemented a treatment regimen that augments the potential antivascular effects of chemotherapy, that is devoid of obvious toxic side effects, and that obstructs the development of drug resistance by tumor cells. Xenografts of 2 independent neuroblastoma cell lines were subjected to either continuous treatment with low doses of vinblastine, a monoclonal neutralizing antibody (DC101) targeting the flk-1/KDR (type 2) receptor for VEGF, or both agents together. The rationale for this combination was that any antivascular effects of the low-dose chemotherapy would be selectively enhanced in cells of newly formed vessels when survival signals mediated by VEGF are blocked. Both DC101 and low-dose vinblastine treatment individually resulted in significant but transient xenograft regression, diminished tumor vascularity, and direct inhibition of angiogenesis. Remarkably, the combination therapy resulted in full and sustained regressions of large established tumors, without an ensuing increase in host toxicity or any signs of acquired drug resistance during the course of treatment, which lasted for >6 months. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org. J. Clin. Invest. 105:R15–R24 (2000).

Targeted disruption of the class B scavenger receptor CD36 protects against atherosclerotic lesion development in mice

Abstract: Macrophage scavenger receptors have been implicated as key players in the pathogenesis of atherosclerosis. To assess the role of the class B scavenger receptor CD36 in atherogenesis, we crossed a CD36-null strain with the atherogenic apo E–null strain and quantified lesion development. There was a 76.5% decrease in aortic tree lesion area (Western diet) and a 45% decrease in aortic sinus lesion area (normal chow) in the CD36-apo E double-null mice when compared with controls, despite alterations in lipoprotein profiles that often correlate with increased atherogenicity. Macrophages derived from CD36-apo E double-null mice bound and internalized more than 60% less copper-oxidized LDL and LDL modified by monocyte-generated reactive nitrogen species. A similar inhibition of in vitro lipid accumulation and foam cell formation after exposure to these ligands was seen. These results support a major role for CD36 in atherosclerotic lesion development in vivo and suggest that blockade of CD36 can be protective even in more extreme proatherogenic circumstances.

Transplanted cord blood–derived endothelial precursor cells augment postnatal neovascularization

Abstract: Endothelial precursor cells (EPCs) have been identified in adult peripheral blood We examined whether EPCs could be isolated from umbilical cord blood, a rich source for hematopoietic progenitors, and whether in vivo transplantation of EPCs could modulate postnatal neovascularization Numerous cell clusters, spindle-shaped and attaching (AT) cells, and cord-like structures developed from culture of cord blood mononuclear cells (MNCs) Fluorescence-trace experiments revealed that cell clusters, AT cells, and cord-like structures predominantly were derived from CD34-positive MNCs (MNC(CD34+)) AT cells and cell clusters could be generated more efficiently from cord blood MNCs than from adult peripheral blood MNCs AT cells incorporated acetylated-LDL, released nitric oxide, and expressed KDR, VE-cadherin, CD31, and von Willebrand factor but not CD45 Locally transplanted AT cells survived and participated in capillary networks in the ischemic tissues of immunodeficient nude rats in vivo AT cells thus had multiple endothelial phenotypes and were defined as a major population of EPCs Furthermore, laser Doppler and immunohistochemical analyses revealed that EPC transplantation quantitatively augmented neovascularization and blood flow in the ischemic hindlimb In conclusion, umbilical cord blood is a valuable source of EPCs, and transplantation of cord blood-derived EPCs represents a promising strategy for modulating postnatal neovascularization

Signaling pathways in insulin action: Molecular targets of insulin resistance

Abstract: Insulin is the most potent anabolic hormone known and is essential for appropriate tissue development, growth, and maintenance of whole-body glucose homeostasis. This hormone is secreted by the β cells of the pancreatic islets of Langerhans in response to increased circulating levels of glucose and amino acids after a meal. Insulin regulates glucose homeostasis at many sites, reducing hepatic glucose output (via decreased gluconeogenesis and glycogenolysis) and increasing the rate of glucose uptake, primarily into striated muscle and adipose tissue. In muscle and fat cells, the clearance of circulating glucose depends on the insulin-stimulated translocation of the glucose transporter GLUT4 isoform to the cell surface (see Shulman, this Perspective series, ref. 1). Insulin also profoundly affects lipid metabolism, increasing lipid synthesis in liver and fat cells, and attenuating fatty acid release from triglycerides in fat and muscle. Insulin resistance occurs when normal circulating concentrations of the hormone are insufficient to regulate these processes appropriately. Thus, by definition, insulin resistance is a defect in signal transduction. The signaling mechanisms involved in the various biologic responses to insulin remain somewhat elusive, but recent progress has shed light on a few pathways that are critical for its regulation of glucose and lipid metabolism. Although insulin affects such diverse processes as cellular growth, differentiation, apoptosis, and lipid, protein, and glucose synthesis and breakdown, we focus here on the regulation of glucose transport as the rate-limiting step in glucose utilization and storage.

JM2, encoding a fork head–related protein, is mutated in X-linked autoimmunity–allergic disregulation syndrome

Abstract: X-linked autoimmunity–allergic disregulation syndrome (XLAAD) is an X-linked recessive immunological disorder characterized by multisystem autoimmunity, particularly early-onset type 1 diabetes mellitus, associated with manifestations of severe atopy including eczema, food allergy, and eosinophilic inflammation. Consistent with the allergic phenotype, analysis of two kindreds with XLAAD revealed marked skewing of patient T lymphocytes toward the Th2 phenotype. Using a positional-candidate approach, we have identified in both kindreds mutations in JM2, a gene on Xp11.23 that encodes a fork head domain–containing protein. One point mutation at a splice junction site results in transcripts that encode a truncated protein lacking the fork head homology domain. The other mutation involves an in-frame, 3-bp deletion that is predicted to impair the function of a leucine zipper dimerization domain. Our results point to a critical role for JM2 in self tolerance and Th cell differentiation. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org. J. Clin. Invest. 106:R75–R81 (2000).

Lessons learned from the development of an Abl tyrosine kinase inhibitor for chronic myelogenous leukemia

Abstract: Protein kinases are a large family of homologous proteins comprising 2 major subfamilies, the protein serine/threonine kinases and protein tyrosine kinases (PTKs). Protein kinases function as components of signal transduction pathways, playing a central role in diverse biological processes such as control of cell growth, metabolism, differentiation, and apoptosis. The development of selective protein kinase inhibitors that can block or modulate diseases with abnormalities in these signaling pathways is considered a promising approach for drug development. Because of their deregulation in human cancers, Bcr-Abl, EGFR, HER2, and protein kinase C (PKC), were among the first protein kinases considered as targets for the development of selective inhibitors. Subsequently, as protein kinases have been implicated in more human cancers (1), drug-discovery efforts have been extended and several first-generation small-molecule inhibitors are now in various stages of development. A selection of these agents is shown in Table ​Table11. Table 1 Selected small-molecule ATP-competitive protein kinase inhibitors in development Based on its clear disease association, we saw the Bcr-Abl tyrosine kinase as an ideal target for validating the clinical utility of protein kinase inhibitors. Here, we discuss our experience in the preclinical and clinical development of a Bcr-Abl inhibitor as a therapeutic agent for chronic myelogenous leukemia (CML), and we consider how this experience and other recent advances in the field could contribute to drug development for other diseases.

Two defects contribute to hypothalamic leptin resistance in mice with diet-induced obesity

Abstract: Obesity in humans and in rodents is usually associated with high circulating leptin levels and leptin resistance. To examine the molecular basis for leptin resistance, we determined the ability of leptin to induce hypothalamic STAT3 (signal transducer and activator of transcription) signaling in C57BL/6J mice fed either low-fat or high-fat diets. In mice fed the low-fat diet, leptin activated STAT3 signaling when administered via the intraperitoneal (ip) or the intracerebroventricular (icv) route, with the half-maximal dose being 30-fold less when given by the icv route. The high-fat diet increased body-weight gain and plasma leptin levels. After 4 weeks on the diet, hypothalamic STAT3 signaling after ip leptin administration was equivalent in both diet groups. In contrast, peripherally administered leptin was completely unable to activate hypothalamic STAT3 signaling, as measured by gel shift assay after 15 weeks of high-fat diet. Despite the absence of detectable signaling after peripheral leptin at 15 weeks, the mice fed the high-fat diet retained the capacity to respond to icv leptin, although the magnitude of STAT3 activation was substantially reduced. These results suggest that leptin resistance induced by a high-fat diet evolves during the course of the diet and has at least two independent causes: an apparent defect in access to sites of action in the hypothalamus that markedly limits the ability of peripheral leptin to activate hypothalamic STAT signaling, and an intracellular signaling defect in leptin-responsive hypothalamic neurons that lies upstream of STAT3 activation.

Melanocortin-4 receptor mutations are a frequent and heterogeneous cause of morbid obesity

Abstract: By integrating an agonist satiety signal, provided by alpha–melanocyte-stimulating hormone (α-MSH), and an antagonist signal, provided by agouti-related protein (AGRP), the melanocortin-4 receptor (MC4-R) is a key element in the hypothalamic control of food intake. Inactivation of the gene encoding this G protein–coupled receptor causes obesity in mice. In humans, frameshift mutations in MC4-R cause an early-onset dominant form of obesity in two families. In this study we find a high frequency (4%) of rare heterozygous MC4-R mutations in a large population of morbidly obese patients. No such mutations were found in controls. By analyzing the phenotypes of the probands carrying these mutations, we demonstrate that these patients display a common, nonsyndromic form of obesity. Interestingly, functional analysis of the mutant receptors indicates that obesity-associated defects in MC4-R range from loss of function to constitutive activation. Transmission of these mutations in the families of the carriers indicates a variable expressivity that is not related to the functional severity of the mutations. This variable expressivity of MC4-R–associated obesity is not due to variations in genes for α-MSH or AGRP. Taken together, these results demonstrate that MC4-R mutations are a frequent but heterogeneous genetic cause of morbid obesity.

Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme

Abstract: We identified hyaluronan synthase-2 (Has2) as a likely source of hyaluronan (HA) during embryonic development, and we used gene targeting to study its function in vivo. Has2(-/-) embryos lack HA, exhibit severe cardiac and vascular abnormalities, and die during midgestation (E9.5-10). Heart explants from Has2(-/-) embryos lack the characteristic transformation of cardiac endothelial cells into mesenchyme, an essential developmental event that depends on receptor-mediated intracellular signaling. This defect is reproduced by expression of a dominant-negative Ras in wild-type heart explants, and is reversed in Has2(-/-) explants by gene rescue, by administering exogenous HA, or by expressing activated Ras. Conversely, transformation in Has2(-/-) explants mediated by exogenous HA is inhibited by dominant-negative Ras. Collectively, our results demonstrate the importance of HA in mammalian embryogenesis and the pivotal role of Has2 during mammalian development. They also reveal a previously unrecognized pathway for cell migration and invasion that is HA-dependent and involves Ras activation.

Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms

Abstract: Abdominal aortic aneurysms represent a life-threatening condition characterized by chronic inflammation, destructive remodeling of the extracellular matrix, and increased local expression of matrix metalloproteinases (MMPs). Both 92-kD gelatinase (MMP-9) and macrophage elastase (MMP-12) have been implicated in this disease, but it is not known if either is necessary in aneurysmal degeneration. We show here that transient elastase perfusion of the mouse aorta results in delayed aneurysm development that is temporally associated with transmural mononuclear inflammation, increased local production of several elastolytic MMPs, and progressive destruction of the elastic lamellae. Elastase-induced aneurysmal degeneration was suppressed by treatment with a nonselective MMP inhibitor (doxycycline) and by targeted gene disruption of MMP-9, but not by isolated deficiency of MMP-12. Bone marrow transplantation from wild-type mice prevented the aneurysm-resistant phenotype in MMP-9-deficient animals, and wild-type mice acquired aneurysm resistance after transplantation from MMP-9-deficient donors. These results demonstrate that inflammatory cell expression of MMP-9 plays a critical role in an experimental model of aortic aneurysm disease, suggesting that therapeutic strategies targeting MMP-9 may limit the growth of small abdominal aortic aneurysms.

Peroxisome proliferator–activated receptor γ ligands inhibit development of atherosclerosis in LDL receptor–deficient mice

Abstract: The peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that regulates fat-cell development and glucose homeostasis and is the molecular target of a class of insulin-sensitizing agents used for the management of type 2 diabetes mellitus. PPARgamma is highly expressed in macrophage foam cells of atherosclerotic lesions and has been demonstrated in cultured macrophages to both positively and negatively regulate genes implicated in the development of atherosclerosis. We report here that the PPARgamma-specific agonists rosiglitazone and GW7845 strongly inhibited the development of atherosclerosis in LDL receptor-deficient male mice, despite increased expression of the CD36 scavenger receptor in the arterial wall. The antiatherogenic effect in male mice was correlated with improved insulin sensitivity and decreased tissue expression of TNF-alpha and gelatinase B, indicating both systemic and local actions of PPARgamma. These findings suggest that PPARgamma agonists may exert antiatherogenic effects in diabetic patients and provide impetus for efforts to develop PPARgamma ligands that separate proatherogenic activities from antidiabetic and antiatherogenic activities.

Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction

Abstract: Matrix metalloproteinase-9 (MMP-9) is prominently overexpressed after myocardial infarction (MI). We tested the hypothesis that mice with targeted deletion of MMP9 have less left ventricular (LV) dilation after experimental MI than do sibling wild-type (WT) mice. Animals that survived ligation of the left coronary artery underwent echocardiographic studies after MI; all analyses were performed without knowledge of mouse genotype. By day 8, MMP9 knockout (KO) mice had significantly smaller increases in end-diastolic and end-systolic ventricular dimensions at both midpapillary and apical levels, compared with infarcted WT mice; these differences persisted at 15 days after MI. MMP-9 KO mice had less collagen accumulation in the infarcted area than did WT mice, and they showed enhanced expression of MMP-2, MMP-13, and TIMP-1 and a reduced number of macrophages. We conclude that targeted deletion of the MMP9 gene attenuates LV dilation after experimental MI in mice. The decrease in collagen accumulation and the enhanced expression of other MMPs suggest that MMP-9 plays a prominent role in extracellular matrix remodeling after MI.

Less is more, regularly: metronomic dosing of cytotoxic drugs can target tumor angiogenesis in mice

Abstract: Chemotherapeutic drugs, long the mainstay of cancer treatment, cause DNA damage and disrupt DNA replication in proliferating cells. Drug regimens have been designed to kill as many tumor cells as possible by treating with “maximum tolerated doses” (MTDs) of these cytotoxic agents. Side effects such as neurotoxicity and damage to proliferating cells in healthy tissues pose serious constraints on the use of chemotherapy. In an effort to balance toxicity with efficacy, a conventional dosing schedule calls for episodic application of a cytotoxic drug at or near the MTD, followed by periods of rest to allow normal tissues to recover. Many such chemotherapy regimens are initially efficacious, resulting in tumor regression or stabilization and prolonged survival. In rare cases, cures are achieved. In general, however, responses are short-lived, with relapses often marked by aggressive cancers that are resistant to the cytotoxic drug. Furthermore, the standard MTD regimen as a rule seriously impairs quality of life. The harsh side effects and the ultimate failures of most chemotherapies have fueled broad investigation of alternatives, including drugs that target not the transformed tumor cells themselves, but rather a genetically stable constituent cell type of tumors, the endothelial cells that form blood vessels. Angiogenesis, the process by which new blood vessels are formed, is a hallmark capability of cancer (1); a compelling body of evidence argues that tumor growth depends on the vasculature, and, in particular, on continuing angiogenesis (2, 3). More than two dozen new drugs that are in or soon to enter clinical trials appear to interfere with tumor angiogenesis (3, 4); there is considerable anticipation about their benefits in treating cancer. Now, two studies suggest a potentially complementary strategy of rescheduling the administration of classical cytotoxic drugs in order to target tumor endothelial cells. It is well established that tumor-associated endothelial cells proliferate during chronic angiogenesis in tumors, albeit at lower frequencies than the tumor cells themselves. Apparently because of their lower rate of cell division, replication of these endothelial cells is only weakly disrupted by the episodic regimens of standard chemotherapeutic protocols. In these two new studies, however, cytotoxic drugs were administered routinely, to target the slowly proliferating tumor endothelial cells and abrogate their apparent capability to repair and recover during the usual rest periods. Both groups worked with mice bearing subcutaneous tumors, and each presents data suggesting that “metronomic” dosing regimens—either continuous infusion or frequent administration without extended rest periods—could have real value in the clinic. Both also demonstrated combinatorial effects of such altered cytotoxic drug regimens with newer, more specific angiogenesis inhibitors.

Dominant and recessive inheritance of morbid obesity associated with melanocortin 4 receptor deficiency

Abstract: Over 20 severely obese subjects in 11 independent kindreds have been reported to have pathogenic heterozygous mutations in the gene encoding the melanocortin 4 receptor (MC4R), making this the most common known monogenic cause of human obesity. To date, the detailed clinical phenotype of this dominantly inherited disorder has not been defined, and no homozygous subjects have been described. We determined the nucleotide sequence of the entire coding region of the MC4R gene in 243 subjects with severe, early-onset obesity. A novel two-base pair GT insertion in codon 279 was found in two unrelated subjects, and four novel missense mutations, N62S, R165Q, V253I, C271Y, and one mutation (T112M) reported previously were found in five subjects. N62S was found in homozygous form in five children with severe obesity from a consanguineous pedigree. All four heterozygous carriers were nonobese. Several features of the phenotype, e.g. hyperphagia, tendency toward tall stature, hyperinsulinemia, and preserved reproductive function, closely resemble those reported previously in Mc4r knock-out mice. In addition, a marked increase in bone mineral density was seen in all affected subjects. In transient transfection assays, the N62S mutant receptor showed a responsiveness to alphaMSH that was intermediate between the wild-type receptor and mutant receptors carrying nonsense and missense mutations associated with dominantly inherited obesity. Thus MC4R mutations result in a syndrome of hyperphagic obesity in humans that can present with either dominant or recessive patterns of inheritance.

Marrow stromal stem cells

Abstract: Multipotential marrow stromal stem cells were known as early as 1968 (1) through the work of Friedenstein and his coworkers, who established that cells that are adherent, clonogenic, nonphagocytic, and fibroblastic in habit (defined as colony-forming units–fibroblastic; CFU-Fs) can be isolated from the bone marrow stroma of postnatal organisms. CFU-Fs, as these investigators found, can give rise under appropriate experimental conditions to a broad spectrum of fully differentiated connective tissues, including cartilage, bone, adipose tissue, fibrous tissue, and myelosupportive stroma (2, 3). Evidence for the physiological relevance of the stromal system and stem cells rests primarily on the in vivo transplantation of marrow stromal cell strains obtained from marrow cell suspensions and expanded in culture. Transplantation of such cells in open systems, such as the space under the kidney capsule, results in the generation of a chimeric ossicle, that is, a structure replicating the histology and architecture of a miniature bone and comprising tissues of both donor and host origin. In these systems, bone trabeculae and cortices, myelosupportive stroma, and adipocytes are of donor origin, whereas the hematopoietic cells that colonize the ossicle and reach full maturity within it are of host origin (4). This outcome can be viewed as the mirror image of bone marrow transplantation, in which host stromal cells provide the structures within which donor cells undergo hematopoiesis. In addition to transplantation in open systems, stromal cell strains can also be transplanted in diffusion chambers that exclude the immigration of host hematopoietic cells into the forming stromal tissues. Under these conditions, an array of differentiated connective tissues — cartilage, bone, fibrous tissue, and adipocytes — develops, all of donor origin (3). In the nonvascularized diffusion chambers, cartilage is more frequently observed than in open transplants and is regularly distributed at sites of predicted low oxygen tension. This principle is reflected in current micromass culturing techniques for obtaining cartilage formation from stromal cells in vitro (5). Cell strains derived from the ex vivo expansion of a single clone (i.e., the progeny of a single CFU-F) are endowed with the same multipotentiality under the same or similar experimental conditions. Thus, a single CFU-F can give rise to ossicles identical to those generated by transplanted nonclonal stromal cell strains, which may include cells of multiple differentiated phenotypes (6). Based on such observations, Friedenstein, Owen, and others developed the concept that cartilage, fat, bone, and other connective tissues derive from a common ancestor, the stromal stem cell. Their studies also established that the stromal stem cell persists within the bone marrow of postnatal and even adult organisms. However, remarkable differences are observed between individual CFU-Fs. Cell morphology and rates of proliferation vary dramatically, as does the ability to form multilayer or nodular structures. Expression of various markers of the osteoblastic, chondrogenic, and adipogenic phenotypes is variable not only between different cell strains, but also within a cell strain, as a function of time in culture. Furthermore, upon transplantation, some CFU-Fs form bone and support hematopoiesis and adipogenesis, some only form bone, while others form only connective tissue (6). To date, no clear-cut phenotypic characteristics have been identified that allow CFU-F subsets to be isolated with predictably broad or restricted potential. Recent attempts, employing monumental numbers of putative markers to purify the true marrow stromal stem cell (inappropriately termed the “mesenchymal stem cell”) from a heterogeneous population of adherent stromal cells, have identified cells that are neither indefinitely self-renewing nor homogeneously multipotential (7). These mesenchymal stem cells, although supposedly purified, reproduce all of the known virtues and vices of the marrow CFU-F population as a whole, as known from Friedenstein’s studies and others’, except that these cells are obtained with considerably lower efficiency than with the earlier protocols. Ironically, the rediscovery of the widely known properties of marrow stromal cells in 1999 was celebrated in the scientific and lay press as the happy product of an extraordinary and successful hunt.

Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide

Abstract: Lipopolysaccharide (LPS) is the main inducer of shock and death in Gram-negative sepsis. Recent evidence suggests that LPS-induced signal transduction begins with CD14-mediated activation of 1 or more Toll-like receptors (TLRs). The lipid A analogues lipid IVa and Rhodobacter sphaeroides lipid A (RSLA) exhibit an uncommon species-specific pharmacology. Both compounds inhibit the effects of LPS in human cells but display LPS-mimetic activity in hamster cells. We transfected human TLR4 or human TLR2 into hamster fibroblasts to determine if either of these LPS signal transducers is responsible for the species-specific pharmacology. RSLA and lipid IVa strongly induced NF-kappaB activity and IL-6 release in Chinese hamster ovary fibroblasts expressing CD14 (CHO/CD14), but these compounds antagonized LPS antagonists in CHO/CD14 fibroblasts that overexpressed human TLR4. No such antagonism occurred in cells overexpressing human TLR2. We cloned TLR4 from hamster macrophages and found that human THP-1 cells expressing the hamster TLR4 responded to lipid IVa as an LPS mimetic, as if they were hamster in origin. Hence, cells heterologously overexpressing TLR4 from different species acquired a pharmacological phenotype with respect to recognition of lipid A substructures that corresponded to the species from which the TLR4 transgene originated. These data suggest that TLR4 is the central lipid A-recognition protein in the LPS receptor complex.

Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in mice

Abstract: The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several endothelium-derived relaxing factors, such as prostacyclin, nitric oxide (NO), and the previously unidentified endothelium-derived hyperpolarizing factor (EDHF). In this study, we examined our hypothesis that hydrogen peroxide (H(2)O(2)) derived from endothelial NO synthase (eNOS) is an EDHF. EDHF-mediated relaxation and hyperpolarization in response to acetylcholine (ACh) were markedly attenuated in small mesenteric arteries from eNOS knockout (eNOS-KO) mice. In the eNOS-KO mice, vasodilating and hyperpolarizing responses of vascular smooth muscle per se were fairly well preserved, as was the increase in intracellular calcium in endothelial cells in response to ACh. Antihypertensive treatment with hydralazine failed to improve the EDHF-mediated relaxation. Catalase, which dismutates H(2)O(2) to form water and oxygen, inhibited EDHF-mediated relaxation and hyperpolarization, but it did not affect endothelium-independent relaxation following treatment with the K(+) channel opener levcromakalim. Exogenous H(2)O(2) elicited similar relaxation and hyperpolarization in endothelium-stripped arteries. Finally, laser confocal microscopic examination with peroxide-sensitive fluorescence dye demonstrated that the endothelium produced H(2)O(2) upon stimulation by ACh and that the H(2)O(2) production was markedly reduced in eNOS-KO mice. These results indicate that H(2)O(2) is an EDHF in mouse small mesenteric arteries and that eNOS is a major source of the reactive oxygen species.

Cathepsin B contributes to TNF-α–mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c

Abstract: TNF-α–induced apoptosis is thought to involve mediators from acidic vesicles. Cathepsin B (cat B), a lysosomal cysteine protease, has recently been implicated in apoptosis. To determine whether cat B contributes to TNF-α–induced apoptosis, we exposed mouse hepatocytes to the cytokine in vitro and in vivo. Isolated hepatocytes treated with TNF-α in the presence of the transcription inhibitor actinomycin D (AcD) accumulated cat B in their cytosol. Further experiments using cell-free systems indicated that caspase-8 caused release of active cat B from purified lysosomes and that cat B, in turn, increased cytosol-induced release of cytochrome c from mitochondria. Consistent with these observations, the ability of TNF-α/AcD to induce mitochondrial release of cytochrome c, caspase activation, and apoptosis of isolated hepatocytes was markedly diminished in cells from CatB–/– mice. Deletion of the CatB gene resulted in diminished liver injury and enhanced survival after treatment in vivo with TNF-α and an adenovirus construct expressing the IκB superrepressor. Collectively, these observations suggest that caspase-mediated release of cat B from lysosomes enhances mitochondrial release of cytochrome c and subsequent caspase activation in TNF-α–treated hepatocytes.