Features that distinguish tumor vasculatures from normal blood vessels are sought to enable the destruction of preformed tumor vessels. We show that blood vessels in both a xenografted tumor and primary human tumors contain a sizable fraction of immature blood vessels that have not yet recruited periendothelial cells. These immature vessels are selectively obliterated as a consequence of vascular endothelial growth factor (VEGF) withdrawal. In a xenografted glioma, the selective vulnerability of immature vessels to VEGF loss was demonstrated by downregulating VEGF transgene expression using a tetracycline-regulated expression system. In human prostate cancer, the constitutive production of VEGF by the glandular epithelium was suppressed as a consequence of androgen-ablation therapy. VEGF loss led, in turn, to selective apoptosis of endothelial cells in vessels devoid of periendothelial cells. These results suggest that the unique dependence on VEGF of blood vessels lacking periendothelial cells can be exploited to reduce an existing tumor vasculature.
We examined potential mechanisms by which angiotensin subtype-2 (AT2) receptor stimulation induces net fluid absorption and serosal guanosine cyclic 3′,5′-monophosphate (cGMP) formation in the rat jejunum. l-arginine (l-ARG) given intravenously or interstitially enhanced net fluid absorption and cGMP formation, which were completely blocked by the nitric oxide (NO) synthase inhibitor, N-nitro-l-arginine methylester (l-NAME), but not by the specific AT2 receptor antagonist, PD-123319 (PD). Dietary sodium restriction also increased jejunal interstitial fluid cGMP and fluid absorption. Both could be blocked by PD or l-NAME, suggesting that the effects of sodium restriction occur via ANG II at the AT2 receptor. l-ARG–stimulated fluid absorption was blocked by the soluble guanylyl cyclase inhibitor 1-H-[1,2,4]oxadiazolo[4,2-α]quinoxalin-1-one (ODQ). Cyclic GMP–specific phosphodiesterase in the interstitial space decreased extracellular cGMP content and prevented the absorptive effects of l-ARG. Angiotensin II (ANG II) caused an increase in net Na+ and Cl– ion absorption and 22Na+ unidirectional efflux (absorption) from the jejunal loop. In contrast, intraluminal heat-stable enterotoxin of Escherichia coli (STa) increased loop cGMP and fluid secretion that were not blocked by either l-NAME or ODQ. These findings suggest that ANG II acts at the serosal side via AT2 receptors to stimulate cGMP production via soluble guanylyl cyclase activation and absorption through the generation of NO, but that mucosal STa activation of particulate guanylyl cyclase causes secretion independently of NO, thus demonstrating the opposite effects of cGMP in the mucosal and serosal compartments of the jejunum.
Allergic asthma, which is present in as many as 10% of individuals in industrialized nations, is characterized by chronic airway inflammation and hyperreactivity induced by allergen-specific Th2 cells secreting interleukin-4 (IL-4) and IL-5. Because Th1 cells antagonize Th2 cell functions, it has been proposed that immune deviation toward Th1 can protect against asthma and allergies. Using an adoptive transfer system, we assessed the roles of Th1, Th2, and Th0 cells in a mouse model of asthma and examined the capacity of Th1 cells to counterbalance the proasthmatic effects of Th2 cells. Th1, Th2, and Th0 lines were generated from ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic mice and transferred into lymphocyte-deficient, OVA-treated severe combined immunodeficiency (SCID) mice. OVA-specific Th2 and Th0 cells induced significant airway hyperreactivity and inflammation. Surprisingly, Th1 cells did not attenuate Th2 cell–induced airway hyperreactivity and inflammation in either SCID mice or in OVA-immunized immunocompetent BALB/c mice, but rather caused severe airway inflammation. These results indicate that antigen-specific Th1 cells may not protect or prevent Th2-mediated allergic disease, but rather may cause acute lung pathology. These findings have significant implications with regard to current therapeutic goals in asthma and allergy and suggest that conversion of Th2-dominated allergic inflammatory responses into Th1-dominated responses may lead to further problems.
Hyperglycemia can cause vascular dysfunctions by multiple factors including hyperosmolarity, oxidant formation, and protein kinase C (PKC) activation. We have characterized the effect of hyperglycemia on p38 mitogen-activated protein (p38) kinase activation, which can be induced by oxidants, hyperosmolarity, and proinflammatory cytokines, leading to apoptosis, cell growth, and gene regulation. Glucose at 16.5 mM increased p38 kinase activity in a time-dependent manner compared with 5.5 mM in rat aortic smooth muscle cells (SMC). Mannitol activated p38 kinase only at or greater than 22 mM. High glucose levels and a PKC agonist activated p38 kinase, and a PKC inhibitor, GF109203X, prevented its activation. However, p38 kinase activation by mannitol or tumor necrosis factor-α was not inhibited by GF109203X. Changes in PKC isoform distribution after exposure to 16.5 mM glucose in SMC suggested that both PKC-β2 and PKC-δ isoforms were increased. Activities of p38 kinase in PKC-δ– but not PKC-β1–overexpressed SMC were increased compared with control cells. Activation of p38 kinase was also observed and characterized in various vascular cells in culture and aorta from diabetic rats. Thus, moderate hyperglycemia can activate p38 kinase by a PKC-δ isoform–dependent pathway, but glucose at extremely elevated levels can also activate p38 kinase by hyperosmolarity via a PKC-independent pathway.
Breast cancer frequently metastasizes to the skeleton, and the associated bone destruction is mediated by the osteoclast. Growth factors, including transforming growth factor-β (TGF-β), released from bone matrix by the action of osteoclasts, may foster metastatic growth. Because TGF-β inhibits growth of epithelial cells, and carcinoma cells are often defective in TGF-β responses, any role of TGF-β in metastasis is likely to be mediated by effects on the surrounding normal tissue. However, we present evidence that TGF-β promotes breast cancer metastasis by acting directly on the tumor cells. Expression of a dominant–negative mutant (TβRIIΔcyt) of the TGF-β type II receptor rendered the human breast cancer cell line MDA-MB-231 unresponsive to TGF-β. In a murine model of bone metastases, expression of TβRIIΔcyt by MDA-MB-231 resulted in less bone destruction, less tumor with fewer associated osteoclasts, and prolonged survival compared with controls. Reversal of the dominant–negative signaling blockade by expression of a constitutively active TGF-β type I receptor in the breast cancer cells increased tumor production of parathyroid hormone–related protein (PTHrP), enhanced osteolytic bone metastasis, and decreased survival. Transfection of MDA-MB-231 cells that expressed the dominant–negative TβRIIΔcyt with the cDNA for PTHrP resulted in constitutive tumor PTHrP production and accelerated bone metastases. These data demonstrate an important role for TGF-β in the development of breast cancer metastasis to bone, via the TGF-β receptor–mediated signaling pathway in tumor cells, and suggest that the bone destruction is mediated by PTHrP.
The transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) is enriched in liver and adipose tissue and controls the expression of a wide variety of genes coding for important metabolic pathways, including gluconeogenesis and lipid synthesis. To investigate the role of C/EBPβ on glucose homeostasis, we studied mice with a targeted deletion of the gene for C/EBPβ–/– mice. Adult C/EBPβ–/– mice have hypoglycemia after an 18-hour fast, accompanied by lower hepatic glucose production (40% of that of wild-type mice), with no change in plasma insulin and a lower concentration of plasma free fatty acids (FFA). Glucagon infusion during a pancreatic clamp acutely stimulated hepatic glucose production by 38% in wild-type animals, with no change detected in C/EBPβ–/– mice. Unexpectedly, both the basal and glucagon-stimulated hepatic cyclic adenosine monophosphate (cAMP) levels were lower in C/EBPβ–/– mice, indicating an essential role for C/EBPβ in controlling proximal signal transduction. Fasting hypoglycemia was associated with normal levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) gene expression, however net liver glycogenolysis was impaired in C/EBPβ–/– mice. FFA release from isolated adipose tissue in response to epinephrine was 68% lower in C/EBPβ–/– mice than in control animals; however, N6,O2′-dibutyryladenosine (Bt2) cAMP stimulated a twofold increase in FFA release in C/EBPβ–/– compared with no further increase in wild-type mice. Because a deletion in the gene for C/EBPβ reduces blood glucose and circulating FFA, it could be an important therapeutic target for the treatment of non–insulin-dependent diabetes and possibly obesity, based on designing antagonists that decrease C/EBPβ activity.
Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by the complete absence of detectable fibrinogen. Uncontrolled bleeding after birth from the umbilical cord is common, and spontaneous intracerebral bleeding and splenic rupture can occur throughout life. Patients respond well to fibrinogen replacement therapy, either prophylactically or on demand. Because the half-life of infused fibrinogen is essentially normal, the genetic defect is assumed to be at the level of synthesis, but no responsible locus has been identified. Preliminary studies using Southern blotting suggested that no gross structural changes of the fibrinogen genes were present in patients. We report the identification of causative mutations in a nonconsanguineous Swiss family with congenital afibrinogenemia. The four affected male individuals (two brothers and their two first cousins) have homozygous deletions of ∼11 kb of the fibrinogen alpha-chain gene (FGA). Haplotype data suggest that these deletions occurred separately, on three distinct ancestral chromosomes, implying that the FGA region of the fibrinogen locus is susceptible to deletion by a common mechanism. Furthermore, our results demonstrate that humans, like mice, may be born without the capacity to synthesize functional fibrinogen.
Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol levels are associated, respectively, with either increased risk or apparent protective effects for atherothrombosis. The ability of purified LDL and HDL to downregulate thrombin formation, a contributor to atherothrombotic processes, was assessed. Purified HDL, but not LDL, significantly enhanced inactivation of coagulation factor Va by activated protein C (APC) and protein S, and HDL stimulated protein S–dependent proteolytic inactivation of Va by APC, apparently due to cleavage at Arg306 in Va. In normal plasma, added HDL enhanced APC/protein S anticoagulant activity in modified prothrombin-time clotting assays. When the anticoagulant potency of HDL was compared with phospholipid (PL) vesicles of well-defined composition using this assay, HDL appeared qualitatively different from PL vesicles because HDL showed only good anticoagulant activity, whereas PL vesicles were rather procoagulant. When 20 normal plasmas were tested using this clotting assay, apoA-I levels correlated with anticoagulant response to APC/protein S (r = 0.47, P = 0.035), but not with activated partial thromboplastin time–based APC resistance ratios. Because HDL enhances the anticoagulant protein C pathway in vitro, we speculate that HDL may help downregulate thrombin generation in vivo and that this anticoagulant action is one of HDL's beneficial activities.
β3 integrins have been implicated in a wide variety of functions, including platelet aggregation and thrombosis (αIIbβ3) and implantation, placentation, angiogenesis, bone remodeling, and tumor progression (αvβ3). The human bleeding disorder Glanzmann thrombasthenia (GT) can result from defects in the genes for either the αIIb or the β3 subunit. In order to develop a mouse model of this disease and to further studies of hemostasis, thrombosis, and other suggested roles of β3 integrins, we have generated a strain of β3-null mice. The mice are viable and fertile, and show all the cardinal features of GT (defects in platelet aggregation and clot retraction, prolonged bleeding times, and cutaneous and gastrointestinal bleeding). Implantation appears to be unaffected, but placental defects do occur and lead to fetal mortality. Postnatal hemorrhage leads to anemia and reduced survival. These mice will allow analyses of the other suggested functions of β3 integrins and we report that postnatal neovascularization of the retina appears to be β3-integrin–independent, contrary to expectations from inhibition experiments.
A line of mice deficient in vitamin D binding protein (DBP) was generated by targeted mutagenesis to establish a model for analysis of DBP's biological functions in vitamin D metabolism and action. On vitamin D–replete diets, DBP–/– mice had low levels of total serum vitamin D metabolites but were otherwise normal. When maintained on vitamin D–deficient diets for a brief period, the DBP–/–, but not DBP+/+, mice developed secondary hyperparathyroidism and the accompanying bone changes associated with vitamin D deficiency. DBP markedly prolonged the serum half-life of 25(OH)D and less dramatically prolonged the half-life of vitamin D by slowing its hepatic uptake and increasing the efficiency of its conversion to 25(OH)D in the liver. After an overload of vitamin D, DBP–/– mice were unexpectedly less susceptible to hypercalcemia and its toxic effects. Peak steady-state mRNA levels of the vitamin D–dependent calbindin-D9K gene were induced by 1,25(OH)2D more rapidly in the DBP–/– mice. Thus, the role of DBP is to maintain stable serum stores of vitamin D metabolites and modulate the rates of its bioavailability, activation, and end-organ responsiveness. These properties may have evolved to stabilize and maintain serum levels of vitamin D in environments with variable vitamin D availability.
To examine the mechanism by which free fatty acids (FFA) induce insulin resistance in human skeletal muscle, glycogen, glucose-6-phosphate, and intracellular glucose concentrations were measured using carbon-13 and phosphorous-31 nuclear magnetic resonance spectroscopy in seven healthy subjects before and after a hyperinsulinemic-euglycemic clamp following a five-hour infusion of either lipid/heparin or glycerol/heparin. IRS-1–associated phosphatidylinositol 3-kinase (PI 3-kinase) activity was also measured in muscle biopsy samples obtained from seven additional subjects before and after an identical protocol. Rates of insulin stimulated whole-body glucose uptake. Glucose oxidation and muscle glycogen synthesis were 50%–60% lower following the lipid infusion compared with the glycerol infusion and were associated with a ∼90% decrease in the increment in intramuscular glucose-6-phosphate concentration, implying diminished glucose transport or phosphorylation activity. To distinguish between these two possibilities, intracellular glucose concentration was measured and found to be significantly lower in the lipid infusion studies, implying that glucose transport is the rate-controlling step. Insulin stimulation, during the glycerol infusion, resulted in a fourfold increase in PI 3-kinase activity over basal that was abolished during the lipid infusion. Taken together, these data suggest that increased concentrations of plasma FFA induce insulin resistance in humans through inhibition of glucose transport activity; this may be a consequence of decreased IRS-1–associated PI 3-kinase activity.
Proteinase-activated receptor-2 (PAR-2) is a G protein–coupled receptor that is cleaved by trypsin within the NH2-terminus, exposing a tethered ligand that binds and activates the receptor. We examined the secretory effects of trypsin, mediated through PAR-2, on well-differentiated nontransformed dog pancreatic duct epithelial cells (PDEC). Trypsin and activating peptide (AP or SLIGRL-NH2, corresponding to the PAR-2 tethered ligand) stimulated both an 125I– efflux inhibited by Ca2+-activated Cl– channel inhibitors and a 86Rb+ efflux inhibited by a Ca2+-activated K+ channel inhibitor. The reverse peptide (LRGILS-NH2) and inhibited trypsin were inactive. Thrombin had no effect, suggesting absence of PAR-1, PAR-3, or PAR-4. In Ussing chambers, trypsin and AP stimulated a short-circuit current from the basolateral, but not apical, surface of PDEC monolayers. In monolayers permeabilized basolaterally or apically with nystatin, AP activated apical Cl– and basolateral K+ conductances. PAR-2 agonists increased [Ca2+]i in PDEC, and the calcium chelator BAPTA inhibited the secretory effects of AP. PAR-2 expression on dog pancreatic ducts and PDEC was verified by immunofluorescence. Thus, trypsin interacts with basolateral PAR-2 to increase [Ca2+]i and activate ion channels in PDEC. In pancreatitis, when trypsinogen is prematurely activated, PAR-2–mediated ductal secretion may promote clearance of toxins and debris.
Patients with resistance to thyroid hormone (RTH) exhibit elevated thyroid hormone levels and inappropriate thyrotropin (thyroid-stimulating hormone, or TSH) production. The molecular basis of this disorder resides in the dominant inhibition of endogenous thyroid hormone receptors (TRs) by a mutant receptor. To determine the relative contributions of pituitary versus hypothalamic resistance to the dysregulated production of thyroid hormone in these patients, we developed a transgenic mouse model with pituitary-specific expression of a mutant TR (Δ337T). The equivalent mutation in humans is associated with severe generalized RTH. Transgenic mice developed profound pituitary resistance to thyroid hormone, as demonstrated by markedly elevated baseline and non–triodothyronine (T3)-suppressible serum TSH and pituitary TSH-β mRNA. Serum thyroxine (T4) levels were only marginally elevated in transgenic mice and thyrotropin-releasing hormone (TRH) gene expression in the paraventricular hypothalamus was downregulated. After TRH administration, T4 concentrations increased markedly in transgenic, but not in wild-type mice. Transgenic mice rendered hypothyroid exhibited a TSH response that was only 30% of the response observed in wild-type animals. These findings indicate that pituitary expression of this mutant TR impairs both T3-mediated suppression and T3-independent activation of TSH production in vivo. The discordance between basal TSH and T4 levels and the reversal with TRH administration demonstrates that resistance at the level of both the thyrotroph and the hypothalamic TRH neurons are required to elevate thyroid hormone levels in patients with RTH.
Relapsing experimental autoimmune encephalomyelitis (R-EAE) in the SJL mouse is a Th1-mediated autoimmune demyelinating disease model for human multiple sclerosis and is characterized by infiltration of the central nervous system (CNS) by Th1 cells and macrophages. Disease relapses are mediated by T cells specific for endogenous myelin epitopes released during acute disease, reflecting a critical role for epitope spreading in the perpetuation of chronic central CNS pathology. We asked whether blockade of the CD40–CD154 (CD40L) costimulatory pathway could suppress relapses in mice with established R-EAE. Anti-CD154 antibody treatment at either the peak of acute disease or during remission effectively blocked clinical disease progression and CNS inflammation. This treatment blocked Th1 differentiation and effector function rather than expansion of myelin-specific T cells. Although T-cell proliferation and production of interleukin (IL)-2, IL-4, IL-5, and IL-10 were normal, antibody treatment severely inhibited interferon-γ production, myelin peptide–specific delayed-type hypersensitivity responses, and induction of encephalitogenic effector cells. Anti-CD154 antibody treatment also impaired the expression of clinical disease in adoptive recipients of encephalitogenic T cells, suggesting that CD40–CD154 interactions may be involved in directing the CNS migration of these cells and/or in their effector ability to activate CNS macrophages/microglia. Thus, blockade of CD154–CD40 interactions is a promising immunotherapeutic strategy for treatment of ongoing T cell–mediated autoimmune diseases.
Acute hypoxic vasoconstriction and development of hypoxic pulmonary hypertension (PHTN) are unique properties of the pulmonary circulation. The pulmonary endothelium produces vasoactive factors, including nitric oxide (NO), that modify these phenomena. We tested the hypothesis that NO produced by endothelial nitric oxide synthase (eNOS) modulates pulmonary vascular responses to hypoxia using mice with targeted disruption of the eNOS gene (eNOS–/–). Marked PHTN was found in eNOS–/– mice raised in mild hypoxia when compared with either controls or eNOS–/– mice raised in conditions simulating sea level. We found an approximate twofold increase in partially and fully muscularized distal pulmonary arteries in eNOS–/– mice compared with controls. Consistent with vasoconstriction being the primary mechanism of PHTN, however, acute inhalation of 25 ppm NO resulted in normalization of RV pressure in eNOS–/– mice. In addition to studies of eNOS–/– mice, the dose–effect of eNOS was tested using heterozygous eNOS+/– mice. Although the lungs of eNOS+/– mice had 50% of normal eNOS protein, the response to hypoxia was indistinguishable from that of eNOS–/– mice. We conclude that eNOS-derived NO is an important modulator of the pulmonary vascular response to chronic hypoxia and that more than 50% of eNOS expression is required to maintain normal pulmonary vascular tone.
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