Issue published December 1, 1999

S everal problems limit the application of gene transfer to correct the cystic fibrosis (CF) Cl^– transport defect in airway epithelia. These include inefficient transduction with vectors applied to the apical surface, a low rate of division by airway epithelial cells, failure of transgene expression to persist, and immune responses to vectors or vector-encoded proteins. To address these issues, we used a feline immunodeficiency virus–based (FIV-based) vector. FIV vector formulated with a calcium chelator transduced fully differentiated, nondividing human airway epithelia when applied to the apical surface. FIV-based vector encoding the cystic fibrosis transmembrane conductance regulator cDNA corrected the Cl^– transport defect in differentiated CF airway epithelia for the life of the culture (>3 months). When this approach was applied in vivo, FIV vector expressing β-galactosidase transduced 1–14% of adult rabbit airway epithelia. Transduced cells were present in the conducting airways, bronchioles, and alveoli. Importantly, gene expression persisted, and cells with progenitor capacity were targeted. FIV-based lentiviral vectors may be useful for the treatment of genetic lung diseases such as CF.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. 104:R55–R62 (1999).

R egulated expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) plays a role in various physiological processes. To determine in vivo how unbalanced expression of these factors can promote or affect the course of pathologies, we knocked out the mouse gelatinase B gene by replacing the catalytic and zinc-binding domains with an antisense-oriented neomycin resistance gene. Adult gelatinase B–deficient mice and wild-type controls could be induced to develop experimental autoimmune encephalomyelitis (EAE) with similar scores for neurologic disease, blood-brain barrier permeability, and central nervous system histopathology. However, whereas diseased control animals showed necrotizing tail lesions with hyperplasia of osteocartilaginous tissue, adult gelatinase B–deficient mice were resistant to this tail pathology. Gelatinase B–deficient mice younger than 4 weeks of age were significantly less susceptible to the development of EAE than were age matched controls and, even as they aged, they remained resistant to tail lesions. These data illustrate that gelatinase B expression plays a role in the development of the immune system and that, in ontogenesis, the propensity to develop autoimmunity is altered by the absence of this MMP.J. Clin. Invest. 104:1507–1515 (1999).

M issense mutations in fibroblast growth factor receptor 3 (FGFR3) result in several human skeletal dysplasias, including the most common form of dwarfism, achondroplasia. Here we show that a glycine-to-cysteine substitution at position 375 (Gly375Cys) in human FGFR3 causes ligand-independent dimerization and phosphorylation of FGFR3 and that the equivalent substitution at position 369 (Gly369Cys) in mouse FGFR3 causes dwarfism with features mimicking human achondroplasia. Accordingly, homozygous mice were more severely affected than heterozygotes. The resulting mutant mice exhibited macrocephaly and shortened limbs due to retarded endochondral bone growth and premature closure of cranial base synchondroses. Compared with their wild-type littermates, mutant mice growth plates shared an expanded resting zone and narrowed proliferating and hypertrophic zones, which is correlated with the activation of Stat proteins and upregulation of cell-cycle inhibitors. Reduced bone density is accompanied by increased activity of osteoclasts and upregulation of genes that are related to osteoblast differentiation, including osteopontin, osteonectin, and osteocalcin. These data reveal an essential role for FGF/FGFR3 signals in both chondrogenesis and osteogenesis during endochondral ossification.J. Clin. Invest. 104:1517–1525 (1999).

P soriasis is a chronic inflammatory skin disease in which epidermal hyperplasia results from skin infiltration by type I T lymphocytes and release of associated cytokines. A multifunctional cytokine, rhIL-11, modulates macrophage and type I T-lymphocyte function in cell culture and shows anti-inflammatory activity in animal models. We are testing subcutaneous delivery of rhIL-11to patients with psoriasis in a phase 1 open-label dose-escalation clinical trial. Tissue was obtained from lesional and uninvolved skin before and during treatment with rhIL-11 and was examined by histology/immunohistochemistry and quantitative RT-PCR. Expression of over 35 genes was examined in all patients, and multiple genetic markers of psoriasis were identified. Expression of numerous proinflammatory genes was elevated in psoriatic tissue compared with nonlesional skin. Seven of 12 patients responded well to rhIL-11 treatment. Amelioration of disease by rhIL-11, as shown by reduced keratinocyte proliferation and cutaneous inflammation, was associated with decreased expression of products of disease-related genes, including K16, iNOS, IFN-γ, IL-8, IL-12, TNF-α, IL-1β, and CD8, and with increased expression of endogenous IL-11. We believe that this is the first study in humans to indicate that type I cytokines can be selectively suppressed by an exogenous immune-modifying therapy. The study highlights the utility of pharmacogenomic monitoring to track patient responsiveness and to elucidate anti-inflammatory mechanisms.J. Clin. Invest. 104:1527–1537 (1999).

G oblet cells are the major mucus-producing cells of the intestine and are presumed to play an important role in mucosal protection. However, their functional role has not been directly assessed in vivo. In initial studies, a 5′ flanking sequence of the murine intestinal trefoil factor (ITF) gene was found to confer goblet cell–specific expression of a transgene. To assess the role of goblet cells in the intestine, we generated transgenic mice in which ∼60% of goblet cells were ablated by the expression of an attenuated diphtheria toxin (DT) gene driven by the ITF promoter; other cell lineages were unaffected. We administered 2 exogenous agents, dextran sodium sulfate (DSS) and acetic acid, to assess the susceptibility of mITF/DT-A transgenic mice to colonic injury. After oral administration of DSS, 55% of control mice died, whereas DT transgenic mice retained their body weight and less than 5% died. Similarly, 30% of the wild-type mice died after mucosal administration of acetic acid, compared with 3.2% of the transgenic mice. Despite the reduction in goblet-cell number, the total amount of ITF was increased in the mITF/DT-A transgenic mice, indicating inducible compensatory mechanisms. These results suggest that goblet cells contribute to mucosal protection and repair predominantly through production of trefoil peptides.J. Clin. Invest. 104:1539–1547 (1999).

D uring the development of nephrotoxic nephritis (NTN) in the mouse, we find that a variety of chemokines and chemokine receptors are induced: CCR1 (RANTES, MIP-1α), CCR2 (MCP-1), CCR5 (RANTES, MIP-1α, MIP-1β), CXCR2 (MIP-2), and CXCR3 (IP-10). Their timing of expression indicated that CXCR2 and CCR1 are probably important in the neutrophil-dependent heterologous phase of the disease, whereas CCR1, CCR2, CCR5, and CXCR3 accompany the subsequent mononuclear cell infiltration characteristic of autologous disease. We therefore assessed the role of CCR1 in NTN using CCR1^–/– mice. We found that neutrophil accumulation in CCR1^–/– mice was comparable to that in wild-type animals but that renal recruitment of CD4⁺ and CD8⁺ T cells and macrophages increased significantly. Moreover, CCR1^–/– mice developed more severe glomerulonephritis than did controls, with greater proteinuria and blood urea nitrogen, as well as a higher frequency of crescent formation. In addition, CCR1^–/– mice showed enhanced Th1 immune responses, including titers of antigen-specific IgG2a antibody, delayed-type hypersensitivity responses, and production of IFN-γ and TNF-α. Lastly, using recombinant proteins and transfected cells that overexpressed CCR1, we demonstrated that MIP-1α, but not RANTES, bound CCR1 and induced cell chemotaxis. Thus, rather than simply promoting leukocyte recruitment during NTN, CCR1 expression profoundly alters the effector phase of glomerulonephritis. Therapeutic targeting of chemokine receptors may, on occasion, exacerbate underlying disease.J. Clin. Invest. 104:1549–1557 (1999).

m Ab 5-1-6 identifies an antigen on rat podocyte slit-diaphragms and induces severe proteinuria when injected into rats. Nephrin, an Ig-like transmembrane protein that is mutated in congenital nephrotic syndrome of the Finnish type, has been localized to the slit-diaphragm on human podocytes. Here we document that the mAb 5-1-6 antigen is rat nephrin. After incubation of rat glomeruli with this mAb, the antibody/antigen complex was chemically cross-linked, extracted, and immunoprecipitated, prior to Western analysis. By mass spectrometry and 2D gel electrophoresis, we identified several peptides with complete identity to human nephrin. In addition, the 185-kDa protein immunoprecipitated by mAb 5-1-6 from rat glomerular extracts reacts with a rabbit anti-mouse nephrin antibody. Finally, nephrin and the mAb 5-1-6 antigen have identical glomerular localization patterns on immunofluorescence of rat kidney. These results demonstrate that the nephritogenic mAb 5-1-6 identifies the extracellular domain of nephrin, thereby documenting the importance of the slit-diaphragm and its component, nephrin, in the regulation of glomerular permselectivity.J. Clin. Invest. 104:1559–1566 (1999).

H eterozygous mutations in NKX2.5, a homeobox transcription factor, were reported to cause secundum atrial septal defects and result in atrioventricular (AV) conduction block during postnatal life. To further characterize the role of NKX2.5 in cardiac morphogenesis, we sought additional mutations in groups of probands with cardiac anomalies and first-degree AV block, idiopathic AV block, or tetralogy of Fallot. We identified 7 novel mutations by sequence analysis of the NKX2.5-coding region in 26 individuals. Associated phenotypes included AV block, which was the primary manifestation of cardiac disease in nearly a quarter of affected individuals, as well as atrial septal defect and ventricular septal defect. Ventricular septal defect was associated with tetralogy of Fallot or double-outlet right ventricle in 3 individuals. Ebstein’s anomaly and other tricuspid valve abnormalities were also present. Mutations in human NKX2.5 cause a variety of cardiac anomalies and may account for a clinically significant portion of tetralogy of Fallot and idiopathic AV block. The coinheritance of NKX2.5 mutations with various congenital heart defects suggests that this transcription factor contributes to diverse cardiac developmental pathways.J. Clin. Invest. 104:1567–1573 (1999).

N emaline myopathy (NM) is a rare autosomal dominant skeletal muscle myopathy characterized by severe muscle weakness and the subsequent appearance of nemaline rods within the muscle fibers. Recently, a missense mutation inTPM3, which encodes the slow skeletal α-tropomyosin (αTm), was linked to NM in a large kindred with an autosomal-dominant, childhood-onset form of the disease. We used adenoviral gene transfer to fully differentiated rat adult myocytes in vitro to determine the effects of NM mutant human αTm expression on striated muscle sarcomeric structure and contractile function. The mutant αTm was expressed and incorporated correctly into sarcomeres of adult muscle cells. The primary defect caused by expression of the mutant αTm was a decrease in the sensitivity of contraction to activating Ca²⁺, which could help explain the hypotonia seen in NM. Interestingly, NM mutant αTm expression did not directly result in nemaline rod formation, which suggests that rod formation is secondary to contractile dysfunction and that load-dependent processes are likely involved in nemaline rod formation in vivo.J. Clin. Invest. 104:1575–1581 (1999).

T he von Hippel-Lindau tumor suppressor protein (pVHL) negatively regulates hypoxia-inducible mRNAs such as the mRNA encoding vascular endothelial growth factor (VEGF). This activity has been linked to its ability to form multimeric complexes that contain elongin C, elongin B, and Cul2. To understand this process in greater detail, we performed a series of in vitro binding assays using pVHL, elongin B, and elongin C variants as well as synthetic peptide competitors derived from pVHL or elongin C. A subdomain of elongin C (residues 17–50) was necessary and sufficient for detectable binding to elongin B. In contrast, elongin B residues required for binding to elongin C were not confined to a discrete colinear domain. We found that the pVHL (residues 157–171) is necessary and sufficient for binding to elongin C in vitro and is frequently mutated in families with VHL disease. These mutations preferentially involve residues that directly bind to elongin C and/or alter the conformation of pVHL such that binding to elongin C is at least partially diminished. These results are consistent with the view that diminished binding of pVHL to the elongins plays a causal role in VHL disease.J. Clin. Invest. 104:1583–1591 (1999).

T he regulation of epithelial Na⁺/H⁺ exchangers (NHEs) by hyposmolality is poorly understood. In the renal medullary thick ascending limb (MTAL), transepithelial bicarbonate (HCO₃^–) absorption is mediated by apical membrane Na⁺/H⁺ exchange, attributable to NHE3. In the present study we examined the effects of hyposmolality on apical Na⁺/H⁺ exchange activity and HCO₃^– absorption in the MTAL of the rat. In MTAL perfused in vitro with 25 mM HCO₃^– solutions, decreasing osmolality in the lumen and bath by removal of either mannitol or sodium chloride significantly increased HCO₃^– absorption. The responses to lumen addition of the inhibitors ethylisopropyl amiloride, amiloride, or HOE 694 are consistent with hyposmotic stimulation of apical NHE3 activity and provide no evidence for a role for apical NHE2 in HCO₃^– absorption. Hyposmolality increased apical Na⁺/H⁺ exchange activity over the pH_i range 6.5–7.5 due to an increase in V_max. Pretreatment with either tyrosine kinase inhibitors or with the tyrosine phosphatase inhibitor molybdate completely blocked stimulation of HCO₃^– absorption by hyposmolality. These results demonstrate that hyposmolality increases HCO₃^– absorption in the MTAL through a novel stimulation of apical membrane Na⁺/H⁺ exchange and provide the first evidence that NHE3 is regulated by hyposmotic stress. Stimulation of apical Na⁺/H⁺ exchange activity in renal cells by a decrease in osmolality may contribute to such pathophysiological processes as urine acidification by diuretics, diuretic resistance, and renal sodium retention in edematous states.J. Clin. Invest. 104:1593–1602 (1999).

A pplication of mechanical strain to neonatal rat ventricular myocytes in culture evokes changes in gene expression reminiscent of those that occur with hypertrophy in vivo, such as stimulation of brain natriuretic peptide (BNP) gene expression. Here, we show that a major component of strain-dependent BNP promoter activation results from stimulation of p38 mitogen-activated protein kinase (MAPK) in the cardiac myocyte. Strain increased p38 activity in a time-dependent fashion. The p38 inhibitor SB203580 led to a reduction of approximately 60% in strain-activated human BNP (hBNP) promoter activity. Cotransfection of wild-type p38 increased both basal and strain-dependent promoter activity, while cotransfection with MKK6AL, a dominant-negative inhibitor of p38 MAPK kinase, resulted in partial inhibition of either p38- or strain-activated hBNP promoter activity. p38 MAPK increased hBNP promoter activity through activation of the transcription factor NF-κB. Activation of the hBNP promoter by either p38 or strain was mediated by DNA elements present in the 5′ flanking sequence of the gene. Mechanical strain promoted assembly of NF-κB components on these DNA elements in vitro. Thus, induction of the hBNP promoter by mechanical strain depends, at least in part, on stimulation of p38 and subsequent activation of NF-κB. This activation may play an important role in signaling the increased BNP gene expression that accompanies hemodynamic overload and cardiac hypertrophy in vivo.J. Clin. Invest. 104:1603–1612 (1999).

V EGF is mitogenic, angiogenic, and a potent mediator of vascular permeability. VEGF causes extravasation of plasma protein in skin bioassays and increases hydraulic conductivity in isolated perfused microvessels. Reduced tissue oxygen tension triggers VEGF expression, and increased protein and mRNA levels for VEGF and its receptors (Flt-1, Flk-1/KDR) occur in the ischemic rat brain. Brain edema, provoked in part by enhanced cerebrovascular permeability, is a major complication in central nervous system pathologies, including head trauma and stroke. The role of VEGF in this pathology has remained elusive because of the lack of a suitable experimental antagonist. We used a novel fusion protein, mFlt(1-3)-IgG, which sequesters murine VEGF, to treat mice exposed to transient cortical ischemia followed by reperfusion. Using high-resolution magnetic resonance imaging, we found a significant reduction in volume of the edematous tissue 1 day after onset of ischemia in mice that received mFlt(1-3)-IgG. 8–12 weeks after treatment, measurements of the resultant infarct size revealed a significant sparing of cortical tissue. Regional cerebral blood flow was unaffected by the administration of mFlt(1-3)-IgG. These results demonstrate that antagonism of VEGF reduces ischemia/reperfusion–related brain edema and injury, implicating VEGF in the pathogenesis of stroke and related disorders.J. Clin. Invest. 104:1613–1620 (1999).

P revious investigations revealed low activities of lactate dehydrogenase (LDH) and plasma membrane monocarboxylate transporters (MCT) in the pancreatic β cell. In this study the significance of these characteristics was explored by overexpressing type A LDH (LDH-A) and/or type 1 MCT (MCT-1) in the clonal INS-1 β cells and isolated rat islets. Inducible overexpression of LDH-A resulted in an 87-fold increase in LDH activity in INS-1 cells. Adenovirus-mediated overexpression of MCT-1 increased lactate transport activity 3.7-fold in INS-1 cells. Although overexpression of LDH-A, and/or MCT-1 did not affect glucose-stimulated insulin secretion, LDH-A overexpression resulted in stimulation of insulin secretion even at a low lactate concentration with a concomitant increase in its oxidation in INS-1 cells regardless of MCT-1 co-overexpression. Adenovirus-mediated overexpression of MCT-1 caused an increase in pyruvate oxidation and conferred pyruvate-stimulated insulin release to isolated rat islets. Although lactate did not stimulate insulin secretion from control or MCT-1–overexpressing islets, co-overexpression of LDH-A and MCT-1 evoked lactate-stimulated insulin secretion with a concomitant increase in lactate oxidation in rat islets. These results suggest that low expression of MCT and LDH is requisite to the specificity of glucose in insulin secretion, protecting the organism from undesired hypoglycemic actions of pyruvate and lactate during exercise and other catabolic states.J. Clin. Invest. 104:1621–1629 (1999).

W e examined the effects of upregulation of heme oxygenase-1 (HO-1) in steatotic rat liver models of ex vivo cold ischemia/reperfusion (I/R) injury. In the model of ischemia/isolated perfusion, treatment of genetically obese Zucker rats with the HO-1 inducer cobalt protoporphyrin (CoPP) or with adenoviral HO-1 (Ad-HO-1) significantly improved portal venous blood flow, increased bile production, and decreased hepatocyte injury. Unlike in untreated rats or those pretreated with the HO-1 inhibitor zinc protoporphyrin (ZnPP), upregulation of HO-1 by Western blots correlated with amelioration of histologic features of I/R injury. Adjunctive infusion of ZnPP abrogated the beneficial effects of Ad-HO-1 gene transfer, documenting the direct involvement of HO-1 in protection against I/R injury. Following cold ischemia/isotransplantation, HO-1 overexpression extended animal survival from 40% in untreated controls to about 80% after CoPP or Ad-HO-1 therapy. This effect correlated with preserved hepatic architecture, improved liver function, and depressed infiltration by T cells and macrophages. Hence, CoPP- or gene therapy–induced HO-1 prevented I/R injury in steatotic rat livers. These findings provide the rationale for refined new treatments that should increase the supply of usable donor livers and ultimately improve the overall success of liver transplantation.J. Clin. Invest. 104:1631–1639 (1999).