Theodore M. Danoff

Evidence that fibroblasts derive from epithelium during tissue fibrosis

Interstitial fibroblasts are principal effector cells of organ fibrosis in kidneys, lungs, and liver. While some view fibroblasts in adult tissues as nothing more than primitive mesenchymal cells surviving embryologic development, they differ from mesenchymal cells in their unique expression of fibroblast-specific protein-1 (FSP1). This difference raises questions about their origin. Using bone marrow chimeras and transgenic reporter mice, we show here that interstitial kidney fibroblasts derive from two sources. A small number of FSP1(+), CD34(-) fibroblasts migrate to normal interstitial spaces from bone marrow. More surprisingly, however, FSP1(+) fibroblasts also arise in large numbers by local epithelial-mesenchymal transition (EMT) during renal fibrogenesis. Both populations of fibroblasts express collagen type I and expand by cell division during tissue fibrosis. Our findings suggest that a substantial number of organ fibroblasts appear through a novel reversal in the direction of epithelial cell fate. As a general mechanism, this change in fate highlights the potential plasticity of differentiated cells in adult tissues under pathologic conditions.

The CoLaus study: a population-based study to investigate the epidemiology and genetic determinants of cardiovascular risk factors and metabolic syndrome.

BackgroundCardiovascular diseases and their associated risk factors remain the main cause of mortality in western societies. In order to assess the prevalence of cardiovascular risk factors (CVRFs) in the Caucasian population of Lausanne, Switzerland, we conducted a population-based study (Colaus Study). A secondary aim of the CoLaus study will be to determine new genetic determinants associated with CVRFs.MethodsSingle-center, cross-sectional study including a random sample of 6,188 extensively phenotyped Caucasian subjects (3,251 women and 2,937 men) aged 35 to 75 years living in Lausanne, and genotyped using the 500 K Affymetrix chip technology.ResultsObesity (body mass index ≥ 30 kg/m2), smoking, hypertension (blood pressure ≥ 140/90 mmHg and/or treatment), dyslipidemia (high LDL-cholesterol and/or low HDL-cholesterol and/or high triglyceride levels) and diabetes (fasting plasma glucose ≥ 7 mmol/l and/or treatment) were present in 947 (15.7%), 1673 (27.0%), 2268 (36.7%), 2113 (34.2%) and 407 (6.6%) of the participants, respectively, and the prevalence was higher in men than in women. In both genders, the prevalence of obesity, hypertension and diabetes increased with age.ConclusionThe prevalence of major CVRFs is high in the Lausanne population in particular in men. We anticipate that given its size, the depth of the phenotypic analysis and the availability of dense genome-wide genetic data, the CoLaus Study will be a unique resource to investigate not only the epidemiology of isolated, or aggregated CVRFs like the metabolic syndrome, but can also serve as a discovery set, as well as replication set, to identify novel genes associated with these conditions.

Early role of Fsp1 in epithelial-mesenchymal transformation

A seamless plasticity exists among cells shifting between epithelial and mesenchymal phenotypes during early development and again later, in adult tissues, following wound repair or organ remodeling in response to injury. Fsp1, a gene encoding a fibroblast-specific protein associated with mesenchymal cell morphology and motility, is expressed during epithelial-mesenchymal transformations (EMT) in vivo. In the current study, we identified several cytokines that induce Fsp1 in cultured epithelial cells. A combination of these factors, however, was most efficacious at completing the process of EMT. The optimal combination identified were two of the cytokines classically associated with fibrosis, i.e., transforming growth factor-β1 (TGF-β1) and epidermal growth factor (EGF). To confirm that it was the induction of Fsp1 by these cytokines mediating EMT, we used antisense oligomers to block Fsp1 production and subsequently measured cell motility and markers of EMT phenotype. The antisense oligomers suppressed Fsp1 expression and epithelial transformation; therefore, we conclude that the appearance of Fsp1 is an important early event in the pathway toward EMT.

Activation of Peroxisome Proliferator–Activated Receptor (PPAR)δ Promotes Reversal of Multiple Metabolic Abnormalities, Reduces Oxidative Stress, and Increases Fatty Acid Oxidation in Moderately Obese Men

OBJECTIVE— Pharmacological use of peroxisome proliferator–activated receptor (PPAR)δ agonists and transgenic overexpression of PPARδ in mice suggest amelioration of features of the metabolic syndrome through enhanced fat oxidation in skeletal muscle. We hypothesize a similar mechanism operates in humans. RESEARCH DESIGN AND METHODS— The PPARδ agonist (10 mg o.d. GW501516), a comparator PPARα agonist (20 μg o.d. GW590735), and placebo were given in a double-blind, randomized, three-parallel group, 2-week study to six healthy moderately overweight subjects in each group. Metabolic evaluation was made before and after treatment including liver fat quantification, fasting blood samples, a 6-h meal tolerance test with stable isotope fatty acids, skeletal muscle biopsy for gene expression, and urinary isoprostanes for global oxidative stress. RESULTS— Treatment with GW501516 showed statistically significant reductions in fasting plasma triglycerides (−30%), apolipoprotein B (−26%), LDL cholesterol (−23%), and insulin (−11%), whereas HDL cholesterol was unchanged. A 20% reduction in liver fat content (P < 0.05) and 30% reduction in urinary isoprostanes (P = 0.01) were also observed. Except for a lowering of triglycerides (−30%, P < 0.05), none of these changes were observed in response to GW590735. The relative proportion of exhaled CO2 directly originating from the fat content of the meal was increased (P < 0.05) in response to GW501516, and skeletal muscle expression of carnitine palmitoyl-transferase 1b (CPT1b) was also significantly increased. CONCLUSIONS— The PPARδ agonist GW501516 reverses multiple abnormalities associated with the metabolic syndrome without increasing oxidative stress. The effect is probably caused by increased fat oxidation in skeletal muscle.

CCL5-CCR5 interaction provides antiapoptotic signals for macrophage survival during viral infection

Host defense against viruses probably depends on targeted death of infected host cells and then clearance of cellular corpses by macrophages. For this process to be effective, the macrophage must presumably avoid its own virus-induced death. Here we identify one such mechanism. We show that mice lacking the chemokine Ccl5 are immune compromised to the point of delayed viral clearance, excessive airway inflammation and respiratory death after mouse parainfluenza or human influenza virus infection. Virus-inducible levels of Ccl5 are required to prevent apoptosis of virus-infected mouse macrophages in vivo and mouse and human macrophages ex vivo. The protective effect of Ccl5 requires activation of the Ccr5 chemokine receptor and consequent bilateral activation of Gαi-PI3K-AKT and Gαi-MEK-ERK signaling pathways. The antiapoptotic action of chemokine signaling may therefore allow scavengers to finally stop the host cell-to-cell infectious process.

Susceptibility to anti-glomerular basement membrane disease and Goodpasture syndrome is linked to MHC class II genes and the emergence of T cell-mediated immunity in mice.

We developed a new mouse model of human anti-glomerular basement membrane (GBM) disease to better characterize the genetic determinants of cell-mediated injury. While all major histocompatibility complex (MHC) haplotypes (H-2a, k, s, b, and d) immunized with alpha3 NC1 domains of type IV collagen produce anti-alpha3(IV) NC1 antibodies that cross-react with human Goodpasture [anti-GBM/anti-alpha3(IV) NC1] autoantibodies, only a few strains developed nephritis and lung hemorrhage associated with Goodpasture syndrome. Crescentic glomerulonephritis and lung hemorrhage were MHC-restricted in haplotypes H-2s, b, and d (A beta/A alpha region in H-2s) and associated with the emergence of an IL-12/Th1-like T cell phenotype. Lymphocytes or anti-alpha3(IV) NC1 antibodies from nephritogenic strains transfer disease to syngeneic recipients. However, passive transfer of isogenic alpha3(IV) NC1 antibodies into -/- T cell receptor-deficient mice failed to produce nephritis. Finally, nephritis and its associated IL-12/Th1-like T cell response attenuate in disease-susceptible mice tolerized orally to alpha3(IV) collagen before immunization. Our findings suggest collectively, as a hypothesis, that anti-GBM antibodies in mice only facilitate disease in MHC haplotypes capable of generating nephritogenic lymphocytes with special T cell repertoires.

An orally active calcium-sensing receptor antagonist that transiently increases plasma concentrations of PTH and stimulates bone formation

Daily subcutaneous administration of exogenous parathyroid hormone (PTH) promotes bone formation in patients with osteoporosis. Here we describe two novel, short-acting calcium-sensing receptor antagonists (SB-423562 and its orally bioavailable precursor, SB-423557) that elicit transient PTH release from the parathyroid gland in several preclinical species and in humans. In an ovariectomized rat model of bone loss, daily oral administration of SB-423557 promoted bone formation and improved parameters of bone strength at lumbar spine, proximal tibia and midshaft femur. Chronic administration of SB-423557 did not increase parathyroid cell proliferation in rats. In healthy human volunteers, single doses of intravenous SB-423562 and oral SB-423557 elicited transient elevations of endogenous PTH concentrations in a profile similar to that observed with subcutaneously administered PTH. Both agents were well tolerated in humans. Transient increases in serum calcium, an expected effect of increased parathyroid hormone concentrations, were observed post-dose at the higher doses of SB-423557 studied. These data constitute an early proof of principle in humans and provide the basis for further development of this class of compound as a novel, orally administered bone-forming treatment for osteoporosis.

Multiple Dose Pharmacokinetics of Paroxetine in Children and Adolescents with Major Depressive Disorder or Obsessive–Compulsive Disorder

The current study examined the pharmacokinetics (PK), safety, and tolerability of paroxetine after repeated multiple oral dosing in children and adolescents with major depressive or obsessive–compulsive disorder. In this 6-week, open-label, repeat dose, dose-rising study, 62 patients (27 children and 35 adolescents) were treated with paroxetine 10 mg/day for the first 2 weeks of the study, 20 mg/day for the next 2 weeks, and 30 mg/day for the final 2 weeks. Pharmacokinetic sampling and safety assessments occurred at baseline and subsequently on the final treatment day of each dosing level. Between-patient variability in PK was pronounced at the 10 mg dose level, but markedly reduced at higher doses. A supra-proportional increase in plasma concentrations with increasing dose was evident in both age groups. Data for Cmax and AUC(0−24) indicated that, at each dose level, paroxetine steady-state systemic exposure was higher in children than in adolescents. The differences between age groups, however, diminished with each increasing dose, and were virtually abolished when differences in weight among different age groups were considered. Stepwise regression analysis indicated that both oral clearance and volume of distribution were highly dependent on paroxetine dose, cytochrome P4502D6 genotype, and weight (p<0.0001), but not age or sex. Paroxetine was generally safe and well tolerated in both age groups, with the most frequently observed adverse events being largely consistent with those observed in prior paroxetine studies of adult psychiatric patients. Certain gastrointestinal and behavioral activation events (aggressive reaction and nervousness) were reported more frequently in the youngest age group.

Digoxin immune fab treatment for severe preeclampsia.

We evaluated the efficacy, safety, and biological mechanisms of digoxin immune Fab (DIF) treatment of severe preeclampsia. Fifty-one severe preeclamptic patients were randomized in double-blind fashion to DIF ( N = 24) or placebo ( N = 27) for 48 hours. Primary outcomes were change in creatinine clearance (CrCl) at 24 to 48 hours and antihypertensive drug use. Serum sodium pump inhibition, a sequela of endogenous digitalis-like factors (EDLF), was also assessed. CrCl in DIF subjects was essentially unchanged from baseline versus a decrease with placebo (-3 +/- 10 and -34 +/- 10 mL/min, respectively, P = 0.02). Antihypertensive use was similar between treatments (46 and 52%, respectively, P = 0.7). Serum sodium pump inhibition was decreased with DIF compared with placebo at 24 hours after treatment initiation (least squares mean difference, 19 percentage points, P = 0.03). DIF appeared to be well tolerated. These results suggest DIF prevents a decline in renal function in severe preeclampsia by neutralizing EDLF. Sodium pump inhibition was significantly improved. Further research is warranted.

Comparison of non-digitalis binding properties of digoxin-specific Fabs using direct binding methods

Digibind and DigiFab are commercial formulations of polyclonal, ovine, digoxin-specific Fabs in clinical use for treatment of digoxin intoxication. Of interest for extending its use to other clinical indications, Digibind has also been reported to neutralize the effect of endogenous digoxin-like molecules, including ouabain, that are linked to clinical disorders ranging from preeclampsia to congestive heart failure. Although Digibind and DigiFab are equivalent in their digoxin-binding activity, the antigens used to produce these Fabs are different. We therefore explored, using native (3)H-digoxin and (3)H-ouabain in four different types of solution-phase binding methods, whether they might exhibit different profiles with respect to ouabain and other digoxin-like factors. Consistent with previous results, both Fab preparations bound digoxin with the same affinities and capacities. However, (3)H-ouabain was found to bind with high affinity only to Fab sub-populations present in both products. Interestingly, this sub-population was twice as large for Digibind compared to DigiFab. Competition experiments also showed differences in specificity within Fab sub-populations. Therefore, the equivalence in digoxin-binding activity of the two Fab preparations does not extend to ouabain-binding capacity and Fab specificity, with implications for clinical differentiation between the preparations in treatment of disorders related to control of non-digoxin cardenolides. The existence of a small but perhaps clinically relevant sub-population of antibodies was detected using specific radioligands. This sub-population could not have been detected nor quantified using standard cross-reactivity in an ELISA assay.