Issue published April 15, 1999

H eme oxygenase (HO) catalyzes the oxidation of heme to generate carbon monoxide (CO) and bilirubin. CO increases cellular levels of cGMP, which regulates vascular tone and smooth muscle development. Bilirubin is a potent antioxidant. Hypoxia increases expression of the inducible HO isoform (HO-1) but not the constitutive isoform (HO-2). To determine whether HO-1 affects cellular adaptation to chronic hypoxia in vivo, we generated HO-1 null (HO-1^–/–) mice and subjected them to hypoxia (10% oxygen) for five to seven weeks. Hypoxia caused similar increases in right ventricular systolic pressure in wild-type and HO-1^–/– mice. Although ventricular weight increased in wild-type mice, the increase was greater in HO-1^–/– mice. Similarly, the right ventricles were more dilated in HO-1^–/– mice. After seven weeks of hypoxia, only HO-1^–/– mice developed right ventricular infarcts with organized mural thrombi. No left ventricular infarcts were observed. Lipid peroxidation and oxidative damage occurred in right ventricular cardiomyocytes in HO-1^–/–, but not wild-type, mice. We also detected apoptotic cardiomyocytes surrounding areas of infarcted myocardium by terminal deoxynucleotide transferase–mediated dUTP nick end-labeling (TUNEL) assays. Our data suggest that in the absence of HO-1, cardiomyocytes have a maladaptive response to hypoxia and subsequent pulmonary hypertension.J.Clin. Invest. 103:R23–R29 (1999).

S kin lesions in atopic dermatitis (AD) are characterized by hypertrophy of the dermis and epidermis, infiltration by T cells and eosinophils, and expression of the cytokines IL-4, IL-5, and IFN-γ. The role of these cytokines in the pathogenesis of AD is not known. We took advantage of a recently described murine model of AD elicited by epicutaneous sensitization with ovalbumin (OVA) (1) and of the availability of mice with targeted deletions of the IL-4, IL-5, and IFN-γ cytokine genes to assess the role of these cytokines in this model.OVA-sensitized skin from IL-5^–/– mice had no detectable eosinophils and exhibited decreased epidermal and dermal thickening. Sensitized skin from IL-4^–/– mice displayed normal thickening of the skin layers but had a drastic reduction in eosinophils and a significant increase in infiltrating T cells. These findings were associated with a reduction in eotaxin mRNA and an increase in mRNA for the T-cell chemokines macrophage inflammatory protein-2 (MIP-2), MIP-1β, and RANTES. Sensitized skin from IFN-γ^–/– mice was characterized by reduced dermal thickening.These results suggest that both the T_H2 cytokines IL-4 and IL-5 and the T_H1 cytokine IFN-γ play important roles in the inflammation and hypertrophy of the skin in AD.

R ecent studies suggest that the gene defect in cystic fibrosis (CF) leads to a breach in innate immunity. We describe a novel genetic strategy for reversing the CF-specific defect of antimicrobial activity by transferring a gene encoding a secreted cathelicidin peptide antibiotic into the airway epithelium grown in a human bronchial xenograft model. The airway surface fluid (ASF) from CF xenografts failed to kill Pseudomonas aeruginosa or Staphylococcus aureus. Partial reconstitution of CF transmembrane conductance regulator expression after adenovirus-mediated gene transfer restored the antimicrobial activity of ASF from CF xenografts to normal levels. Exposure of CF xenografts to an adenovirus expressing the human cathelicidin LL-37/hCAP-18 increased levels of this peptide in the ASF three- to fourfold above the normal concentrations, which were equivalent in ASF from CF and normal xenografts before gene transfer. The increase of LL-37 was sufficient to restore bacterial killing to normal levels. The data presented describe an alternative genetic approach to the treatment of CF based on enhanced expression of an endogenous antimicrobial peptide and provide strong evidence that expression of antimicrobial peptides indeed protects against bacterial infection.

G astrin is a peptide hormone involved in the growth of both normal and malignant gastrointestinal tissue. Recent studies suggest that the glycine-extended biosynthetic intermediates mediate many of these trophic effects, but the in vivo relevance of glycine-extended gastrin (G-Gly) has not been tested. We have generated mice (MTI/G-GLY) that overexpress progastrin truncated at glycine-72 to evaluate the trophic effects of G-Gly in an in vivo model. MTI/G-GLY mice have elevated serum and colonic mucosal levels of G-Gly compared with wild-type mice. MTI/G-GLY mice had a 43% increase in colonic mucosal thickness and a 41% increase in the percentage of goblet cells per crypt. MTI/G-GLY mice exhibited increased colonic proliferation compared with wild-type controls, with an expansion of the proliferative zone into the upper third of the colonic crypts. Continuous infusion of G-Gly into gastrin-deficient mice for two weeks also resulted in elevated G-Gly levels, a 10% increase in colonic mucosal thickness, and an 81% increase in colonic proliferation when compared with gastrin-deficient mice that received saline alone. To our knowledge, these studies demonstrate for the first time that G-Gly’s contribute to colonic mucosal proliferation in vivo.

A cute porphyrias are inherited disorders caused by partial deficiency of specific heme biosynthesis enzymes. Clinically, porphyrias are manifested by a neuropsychiatric syndrome that includes peripheral neuropathy. Although much is known about the porphyrias’ enzyme defects and their biochemical consequences, the cause of the neurological manifestations remains unresolved. We have studied porphyric neuropathy in mice with a partial deficiency of porphobilinogen deaminase (PBGD). PBGD-deficient mice (PBGD^–/–) imitate acute porphyria through massive induction of hepatic δ-aminolevulinic acid synthase by drugs such as phenobarbital. Here we show that PBGD^–/– mice develop impairment of motor coordination and muscle weakness. Histologically femoral nerves of PBGD^–/– mice exhibit a marked decrease in large-caliber (>8 μm) axons and ultrastructural changes consistent with primary motor axon degeneration, secondary Schwann cell reactions, and axonal regeneration. These findings resemble those found in studies of affected nerves of patients with acute porphyria and thus provide strong evidence that PBGD deficiency causes degeneration of motor axons without signs of primary demyelination, thereby resolving a long-standing controversy. Interestingly, the neuropathy in PBGD^–/– mice developed chronically and progressively and in the presence of normal or only slightly (twofold) increased plasma and urinary levels of the putative neurotoxic heme precursor δ-aminolevulinic acid. These data suggest that heme deficiency and consequent dysfunction of hemeproteins can cause porphyric neuropathy.

S ystemic lupus erythematosus (SLE) is an autoimmune disease characterized by various autoantibodies that recognize autoantigens displayed on the surface of cells undergoing apoptosis. The genetic contribution to SLE susceptibility has been widely recognized. We previously reported evidence for linkage to SLE of the human chromosome 1q41–q42 region and have now narrowed it from 15 to 5 cM in an extended sample using multipoint linkage analysis. Candidate genes within this region include (a) PARP, poly(ADP-ribose) polymerase, encoding a zinc-finger DNA-binding protein that is involved in DNA repair and apoptosis; (b) TGFB2, encoding a transforming growth factor that regulates cellular interactions and responses; and (c) HLX1, encoding a homeobox protein that may regulate T-cell development. Using a multiallelic, transmission-disequilibrium test (TDT), we found overall skewing of transmission of PARP alleles to affected offspring in 124 families (P = 0.00008), preferential transmission of a PARP allele to affected offspring (P = 0.0003), and lack of transmission to unaffected offspring (P = 0.004). Similar TDT analyses of TGFB2 and HLX1 polymorphisms yielded no evidence for association with SLE. These results suggest that PARP may be (or is close to) the susceptibility gene within the chromosome 1q41–q42 region linked to SLE.

S hear stress, the tangential component of hemodynamic forces, plays an important role in endothelial remodeling. In this study, we investigated the role of Rho family GTPases Cdc42 and Rho in shear stress–induced signal transduction and cytoskeleton reorganization. Our results showed that shear stress induced the translocation of Cdc42 and Rho from cytosol to membrane. Although both Cdc42 and Rho were involved in the shear stress–induced transcription factor AP-1 acting on the 12-O-tetradecanoyl-13-phorbol-acetate–responsive element (TRE), only Cdc42 was sufficient to activate AP-1/TRE. Dominant-negative mutants of Cdc42 and Rho, as well as recombinant C3 exoenzyme, attenuated the shear stress activation of c-Jun NH₂-terminal kinases (JNKs), suggesting that Cdc42 and Rho regulate the shear stress induction of AP-1/TRE activity through JNKs. Shear stress–induced cell alignment and stress fiber formation were inhibited by the dominant-negative mutants of Rho and p160ROCK, but not by the dominant-negative mutant of Cdc42, indicating that the Rho-p160ROCK pathway regulates the cytoskeletal reorganization in response to shear stress.

X -linked hyper-IgM syndrome (XHIM) results from mutations in the gene encoding for CD40 ligand (CD154). Patients with the syndrome suffer from infections with opportunistic pathogens such as Cryptosporidium and Pneumocystis carinii. In this study, we demonstrate that activated T cells from patients with XHIM produce markedly reduced levels of IFN-γ, fail to induce antigen-presenting cells to synthesize IL-12, and induce greatly reduced levels of TNF-α. In addition, we show that the patients’ circulating T lymphocytes of both the CD4⁺ and CD8⁺ subsets contain a markedly reduced antigen-primed population, as determined by CD45RO expression. Finally, we demonstrate that the defects in antigen priming are likely due to the lack of CD154 expression and insufficient costimulation of T cells by CD80/CD86 interactions. Taken together, this study offers a basis for the increased susceptibility of patients with XHIM to certain opportunistic infections.

K idney proximal tubule cells take up Krebs cycle intermediates for metabolic purposes and for secretion of organic anions through dicarboxylate/organic anion exchange. Alteration in reabsorption of citrate is closely related to renal stone formation. The presence of distinct types of sodium-coupled dicarboxylate transporters has been postulated on either side of the polarized epithelial membrane in the kidney proximal tubule. Using a PCR-based approach, we isolated a novel member of the sodium-dependent dicarboxylate/sulfate transporter called SDCT2. SDCT2 is a 600–amino acid residue protein that has 47–48% amino acid identity to SDCT1 and NaDC-1, previously identified in kidney and intestine. Northern analysis gave a single band of 3.3 kb for SDCT2 in kidney, liver, and brain. In situ hybridization revealed that SDCT2 is prominently expressed in kidney proximal tubule S3 segments and in perivenous hepatocytes, consistent with the sites of high-affinity dicarboxylate transport identified based on vesicle studies. A signal was also detected in the meningeal layers of the brain. SDCT2 expressed in Xenopus oocytes mediated sodium-dependent transport of di- and tricarboxylates with substrate preference for succinate rather than citrate, but excluding monocarboxylates. SDCT2, unlike SDCT1, displayed a unique pH dependence for succinate transport (optimal pH 7.5–8.5) and showed a high affinity for dimethylsuccinate, two features characteristic of basolateral transport. These data help to interpret the mechanisms of renal citrate transport, their alteration in pathophysiological conditions, and their role in the elimination of organic anions and therapeutic drugs.

L arge neutral amino acids (LNAAs), including phenylalanine (Phe), compete for transport across the blood-brain barrier (BBB) via the L-type amino acid carrier. Accordingly, elevated plasma Phe impairs brain uptake of other LNAAs in patients with phenylketonuria (PKU). Direct effects of elevated brain Phe and depleted LNAAs are probably major causes for disturbed brain development and function in PKU. Competition for the carrier might conversely be put to use to lower Phe influx when the plasma concentrations of all other LNAAs are increased. This hypothesis was tested by measuring brain Phe in patients with PKU by quantitative ¹H magnetic resonance spectroscopy during an oral Phe challenge with and without additional supplementation with all other LNAAs. Baseline plasma Phe was ∼1,000 μmol/l and brain Phe was ∼250 μmol/l in both series. Without LNAA supplementation, brain Phe increased to ∼400 μmol/l after the oral Phe load. Electroencephalogram (EEG) spectral analysis revealed acutely disturbed brain activity. With concurrent LNAA supplementation, Phe influx was completely blocked and there was no slowing of EEG activity. These results are relevant for further characterization of the LNAA carrier and of the pathophysiology underlying brain dysfunction in PKU and for treatment of patients with PKU, as brain function might be improved by continued LNAA supplementation.

S cleroderma currently affects approximately 75,000–100,000 individuals in the United States. Fibroblasts isolated from lesional skin of scleroderma patients overexpress collagens and other matrix components, and this abnormality is maintained for multiple passages in culture. To understand the molecular basis for matrix gene overexpression, we performed a differential display comparison of fibroblasts from clinically lesional and nonlesional scleroderma skin. The results suggested that protease nexin 1 (PN1), a protease inhibitor, is overexpressed in scleroderma fibroblasts. Northern blot verification showed that lesional and nonlesional scleroderma fibroblasts had three- to five-fold increased levels of PN1 mRNA compared with healthy fibroblasts. Western analysis showed that scleroderma fibroblasts also secreted more PN1. In situ hybridization of skin biopsy specimens demonstrated PN1 expression in the dermis of four out of six scleroderma patients but no PN1 expression in the dermis of six healthy volunteers. Transient or stable overexpression of PN1 in mouse 3T3 fibroblasts increased collagen promoter activity or endogenous collagen transcript levels, respectively. PN1 mutagenized at its active site and antisense PN1 both failed to increase collagen promoter activity. These results suggest that overexpression of enzymatically active PN1 may play a pathogenic role in the development of the scleroderma phenotype.

T riglyceride (TG) enrichment of HDL resulting from cholesteryl ester transfer protein–mediated exchange with TG-rich lipoproteins may enhance the lipolytic transformation and subsequent metabolic clearance of HDL particles in hypertriglyceridemic states. The present study investigates the effect of TG enrichment of HDL on the clearance of HDL-associated apo A-I in humans. HDL was isolated from plasma of six normolipidemic men (mean age: 29.7 ± 2.7 years) in the fasting state and after a five-hour intravenous infusion with a synthetic TG emulsion, Intralipid. Intralipid infusion resulted in a 2.1-fold increase in the TG content of HDL. Each tracer was then whole-labeled with ¹²⁵I or ¹³¹I and injected intravenously into the subject. Apo A-I in TG-enriched HDL was cleared 26% more rapidly than apo A-I in fasting HDL. A strong correlation between the Intralipid-induced increase in the TG content of HDL and the increase in HDL apo A-I fractional catabolic rate reinforced the importance of TG enrichment of HDL in enhancing its metabolic clearance. HDL was separated further into lipoproteins containing apo A-II (LpAI:AII) and those without apo A-II (LpAI). Results revealed that the enhanced clearance of apo A-I from TG-enriched HDL could be largely attributed to differences in the clearance of LpAI but not LpAI:AII. This is, to our knowledge, the first direct demonstration in humans that TG enrichment of HDL enhances the clearance of HDL apo A-I from the circulation. This phenomenon could provide an important mechanism explaining how HDL apo A-I and HDL cholesterol are lowered in hypertriglyceridemic states.

A utoimmune diabetes in nonobese diabetic (NOD) mice results from destruction of pancreatic β cells by T lymphocytes. It is believed that CD8⁺ cytotoxic T lymphocytes (CTLs) effect the initial β-cell insult in diabetes, but the mechanisms remain unclear. Studies of NOD.lpr mice have suggested that disease initiation is a Fas-dependent process, yet perforin-deficient NOD mice rarely develop diabetes despite expressing Fas. Here, we have investigated the role of perforin and Fas in the ability of β cell–reactive CD8⁺ T cells bearing a T-cell receptor (8.3-TCR) that is representative of TCRs used by CD8⁺ CTLs propagated from the earliest insulitic lesions of NOD mice, and that targets an immunodominant peptide/H-2K^d complex on β cells, to effect β-cell damage in vitro and in vivo. In vitro, 8.3-CTLs killed antigenic peptide–pulsed non–β-cell targets via both perforin and Fas, but they killed NOD β cells via Fas exclusively. Perforin-deficient 8.3-TCR–transgenic NOD mice expressing an oligoclonal or monoclonal T-cell repertoire developed diabetes even more frequently than their perforin-competent littermates. These results demonstrate that diabetogenic CD8⁺ CTLs representative of CTLs putatively involved in the initiation of autoimmune diabetes kill β cells in a Fas-dependent and perforin-independent manner.

S erine elastases degrade elastin, stimulate vascular smooth muscle cell migration and proliferation, and are associated with myocardial damage. To evaluate the impact of elastase inhibition on cardiovascular development and disease, transgenic mice were created in which the mouse preproendothelin-1 promoter was used to target elafin overexpression to the cardiovascular system. To distinguish the transgene from endogenous elafin, constructs were made incorporating a FLAG sequence; the COOH-terminus FLAG-tagged elafin construct produced a stable, functionally active gene product and was used to create transgenic mice. Consistent with endothelin expression, abundant elafin mRNA was observed in transgenic F1 embryos (embryonic day 13.5) and in adult transgenic mice heart, trachea, aorta, kidney, lung, and skin, but not in liver, spleen, and intestine. Functional activity of the transgene was confirmed by heightened myocardial elastase inhibitory activity. No tissue abnormalities were detected by light microscopy or elastin content. However, injection of 10 plaque-forming units (PFU) of encephalomyocarditis virus resulted in death within 11 days in 10 out of 12 nontransgenic mice compared with one out of nine transgenic littermates. This reduced mortality was associated with better cardiac function and less myocardial inflammatory damage. Thus, elafin expression may confer a protective advantage in myocarditis and other inflammatory diseases.