A new animal diet based on human Western diet is a robust diet-induced obesity model: comparison to high-fat and cafeteria diets in term of metabolic and gut microbiota disruption.

BACKGROUND/OBJECTIVES: Obesity is a metabolic disorder that predisposes patients to numerous diseases and has become a major global public-health concern. Animal models of diet-induced obesity (DIO) are frequently used to study obesity, but which DIO model most accurately reflects the pathology of human obesity remains unclear. In this study, we designed a diet based on the human Western diet (WD) and compared it with the cafeteria diet (CAF) and high-fat diet (HFD) in order to evaluate which diet most closely mirrors human obesity. METHODS: Wistar rats were fed four different diets (WD, CAF, HFD and a low-fat diet) for 18 weeks. Metabolic parameters and gut microbiota changes were then characterized. RESULTS: Rats fed the four different diets exhibited completely different phenotypes, highlighting the importance of diet selection. This study also revealed that WD most effectively induced obesity and obesity-related disorders, and thus proved to be a robust model of human obesity. Moreover, WD-fed rats developed obesity and obesity-related comorbidities independent of major alterations in gut microbiota composition (dysbiosis), whereas CAF-fed rats developed the greatest dysbiosis independent of obesity. We also characterized gut microbiota after feeding on these four different diets and identified five genera that might be involved in the pathogenesis of obesity. CONCLUSIONS: These data suggest that diet, and not the obese state, was the major driving force behind gut microbiota changes. Moreover, the marked dysbiosis observed in CAF-fed rats might have resulted from the presence of several additives present in the CAF diet, or even a lack of essential vitamins and minerals. Based on our findings, we recommend the use of the prototypic WD (designed here) in DIO models. Conversely, CAF could be used to investigate the effects of excessive consumption of industrially produced and highly processed foods, which are characteristic of Western society.

Preventive strategies in paediatric cardiovascular surgery: impact on surgical site infections and beyond.

BACKGROUND: Surgical management of congenital heart disease (CHD) has increased worldwide, but healthcare-associated infections (HAIs) can threaten these efforts. AIM: To analyse the incidence of HAI, the impact of preventive interventions, and microbiological profiles in a paediatric cardiovascular surgery programme. METHODS: Cohort study including children aged <12 years with CHD who underwent cardiovascular surgery between 2010 and 2021 in Medellín, Colombia (a middle-income setting). Data were collected from medical and laboratory records and infection control programme databases. Impact of various preventive interventions was assessed using a Poisson model. P < 0.05 was considered statistically significant. FINDINGS: A total of 2512 surgeries were analysed. Incidence of surgical site infection (SSI) was 5.9%, followed by central line-associated bloodstream infection (CLABSI; 4.7%), catheter-associated urinary tract infection (CAUTI; 2.2%), and ventilator-associated pneumonia (VAP; 1.4%). Most of the strategies focused on preventing SSI, resulting in a reduction from 9.5% in 2010 to 3.0% in 2021 (P = 0.030). Antibiotic prophylaxis based on patient weight and continuous infusion had an impact on reducing SSI (RR: 0.56; 95% CI: 0.32-0.99). Vacuum-assisted closure (VAC) in clean wounds reduced 100% of infections. No significant risk reduction was observed for other HAI with the implemented interventions. CONCLUSION: Preventive strategies effectively reduced SSI but no other infections, emphasizing the need for targeted approaches to address a broader spectrum of HAI successfully.

In vitro induction of abnormal anaphases by contaminating atmospheric dust from the City of Mexicali, Baja California, Mexico.

Mexicali dust (MD) is a mixture of particles of potassium aluminum silicates (98%) and sodium dioxide (2%) that induces pulmonary damage under experimental conditions, and is capable of inducing in vitro chromosomal alterations in exposed lymphocytes. It has been proposed as an atmospheric contaminant with pathogenic potential. Among the chromosomal alterations observed, numeric alterations were predominant. The present study was designed to evaluate the capacity of MD to induce anaphasic changes in the Balb c 3T3 cell line. Chrysotile asbestos was used as a positive control. MD was found to induce abnormal anaphases, and the percentage of abnormalities increased as the dose increased (27.41% with 20 mg/mL, 29.60% with 40 mg/mL and 37.10% with 80 mg/mL). Multipolar anaphases constituted the most frequent alteration (69.1-78.8%), followed by lagging chromosomes (18.2-29.5%) and anaphasic bridges (1.51-5.9%). The anaphasic alterations induced by MD showed differences in comparison to those observed with asbestos, especially for anaphasic bridges (10.4% vs. 1.51%, p<0.05). The capacity of MD to induce alterations reported in the process of chromosomal disjunction could explain the numeric aberrations reported previously by the authors of this paper. Therefore, these data support that MD could act as a clastogenic agent.

[Cardiovascular pathology and the partial autopsy. The experience at the Instituto Nacional de Cardiología Ignacio Chávez of Mexico]. / La patología cardiovascular y la autopsia parcial. Experiencia en el Instituto Nacional de Cardiología Ignacio Chávez de México.

There is no doubt autopsies are still a powerful source of medical information. At the Instituto Nacional de Cardiología de México 50% of deaths are autopsied. About half are limited cardiothoracic studies. Since no previous evaluation of the utility of the information obtained from those limited necropsies, we decided to test how well they allowed for a good clinical and anatomical correlation of main disease and a cause of death, as well as their potential to respond clinical questions relevant to the case. We analyzed the medical and autopsy records from 50 cases and determined autopsy type (limited or total), age, sex, main disease and cause of death. It was also included a list of questions made by the attending physician who asked for autopsy. Twenty-six cases corresponded to limited autopsies. In 96.1% there was a good anatomical and clinical correlation of main disease. In 69.2% the cause of death had also a good correlation and in 92.3% of the cases the clinician's question were answered appropriately. Based in our results we support the idea that cardiovascular limited autopsies are an alternative way to obtain useful information, when otherwise, total autopsies result expensive or difficult to obtain.

[Antifoaming agent microembolism in patients undergoing extracorporeal circulation. Its frequency in post mortem material and its pathogenic potential in vitro]. / Microembolias de antiespumante en pacientes sometidos a circulación extracorpórea. Frecuencia en material post mortem y su potencial patógeno in vitro.

Antifoam microembolisms in patients that undergo open heart surgery, represent a risk for postoperative complications. We decided to study its frequency in an autopsy population of patients who died after heart surgery. Forty-five patients were selected and histological sections from the kidneys were studied under light microscopy, scanning electron microscopy and X-ray microanalysis. Thirty-six cases (80%) showed microemboli in the glomerular capillaries lumens. There was a positive correlation between the number of particles found and the length of the surgery. Microemboli were composed of an amorphous fraction and a particulated one composed of silicon. In vitro experimentation demonstrated that the particles are capable to induce cell lysis in a dose related manner. They also are susceptible of been phagocitated by macrophages. We conclude that bubble oxygenator are capable to induce microembolisms in a high percentage of the cases studied. Components of the microemboli are cytotoxic. Therefore microembolisms could be participating in the morbidity of patients subjects to cardiopulmonary bypass.

Exposure to inhaled particulate matter activates early markers of oxidative stress, inflammation and unfolded protein response in rat striatum.

To study central nervous system airborne PM related subchronic toxicity, SD male rats were exposed for eight weeks to either coarse (32 µg/m³), fine (178 µg/m³) or ultrafine (107 µg/m³) concentrated PM or filtered air. Different brain regions (olfactory bulb, frontal cortex, striatum and hippocampus), were harvested from the rats following exposure to airborne PM. Subsequently, prooxidant (HO-1 and SOD-2), and inflammatory markers (IL-1ß and TNFα), apoptotic (caspase 3), and unfolded protein response (UPR) markers (XBP-1S and BiP), were also measured using real-time PCR. Activation of nuclear transcription factors Nrf-2 and NF-κB, associated with antioxidant and inflammation processes, respectively, were also analyzed by GSMA. Ultrafine PM increased HO-1 and SOD-2 mRNA levels in the striatum and hippocampus, in the presence of Nrf-2 activation. Also, ultrafine PM activated NF-κB and increased IL-1ß and TNFα in the striatum. Activation of UPR was observed after exposure to coarse PM through the increment of XBP-1S and BiP in the striatum, accompanied by an increase in antioxidant response markers HO-1 and SOD-2. Our results indicate that exposure to different size fractions of PM may induce physiological changes (in a neuroanatomical manner) in the central nervous system (CNS), specifically within the striatum, where inflammation, oxidative stress and UPR signals were effectively activated.

[Early histologic diagnosis of acute myocardial infarct by autofluorescence. Evaluation in humans and experimental animals]. / Diagnostico histológico temprano del infarto agudo del miocardio por autofluorescencia. Una evaluacion en humanos y en animales de experimentación.

The value of an autofluorescence method for the early diagnosis of myocardial infarction was evaluated in humans and experimental animals. The hearts from 15 rats and 18 dogs that had undergone coronary occlusion, and those obtained from 29 human autopsy cases with clinical evidence of myocardial infarction were studied. Routine and frozen sections stained and unstained were observed under fluorescence microscopy. In all the cases, ischemic areas exhibited fibres with a bright yellow autofluorescence. There was some variations in fluorescence intensity from routine stained and unstained to frozen stained and unstained sections. These results demonstrate that the fluorescence method can be used to defect early myocardial ischemic injury. The intracellular alterations induced by ischemia could be responsible for light absorption and thus characteristic autofluorescence.

Morphology surface of a mouse plasmacytoma (LPC-1) showing cyclic resistance to immune lysis.

The murine BALB/c myeloma LPC-1 demonstrates a periodic resistance to lysis by immune mechanisms; this correlates with the production and accumulation of a trypsin-sensitive, single chain glycoprotein of 160 kilodaltons gp160 on the tumor cell surface. Tumor cells obtained 4 days after transplantation are lysed by cytotoxic T lymphocytes whereas ten-day cells are resistant to lysis. The progressive resistance to lysis was correlated with an increasing amount of gp160 on the surface of LPC-1 cells. Cell surface morphology, as determined by scanning electron microscopy, showed that early cells consisted of equal proportions of cells having microvilli or ruffles. The late cell population consisted mainly of cells with microvilli. These microvilli were twice as abundant on late LPC-1 cells as on early cells. Transmission electron microscopy images of late LPC-1 cells suggested an active protein synthesis which correlated with a more intense deposition of ruthenium red and an increasing amount of gp160 on the cell surface.

Differential binding and regulation of platelet-derived growth factor A and B chain isoforms by alpha 2-macroglobulin.

Alpha 2-Macroglobulin (alpha 2M) is a multifunctional secreted glycoprotein that serves as a ubiquitous proteinase inhibitor and as a binding protein for platelet-derived growth factor (PDGF) BB and homologues of PDGF-BB secreted in culture by macrophages. The interaction of alpha 2M with PDGF-A chain molecules has not been addressed. This is a potentially important issue because fibroblasts and smooth muscle cells produce PDGF-AA, whereas macrophages produce mainly PDGF-BB. Recombinant human 125I-PDGF-B chain molecules (AB and BB) bound to plasma-derived, native human, or bovine alpha 2M and trypsin-activated alpha 2M on Superose 6 fast protein liquid chromatography gel filtration and on nondenaturing polyacrylamide gel electrophoresis, whereas 125I-PDGF-AA did not. Similar results were obtained with 125I-PDGF isoforms binding to immobilized bovine alpha 2M and alpha 2M-methylamine. The same differential pattern of unlabeled PDGF isoforms binding to alpha 2M was observed by Western blotting of PDGF. Human lung fibroblasts secreted alpha 2M as measured by Western blotting, and fibroblast-derived alpha 2M possessed the same differential binding pattern for PDGF isoforms as did plasma-derived alpha 2M. The specific binding of PDGF-AB and -BB to these fibroblasts was inhibited by native bovine alpha 2M, although PDGF-AA binding was not affected. Native alpha 2M preferentially blocked fibroblast chemotaxis to the PDGF-B chain dimers. These data suggest that only PDGF-B chain dimers, such as those produced by macrophages or released from platelets, are regulated by alpha 2M and that PDGF-AA produced by fibroblasts and smooth muscle cells is not controlled by this cytokine-binding protein.

[Autopsy: reflection of intrahospital mortality]. / La autopsia, espejo de la mortalidad Intrahospitalaria.

Autopsies have long been viewed as a biased source of information with regard to the mortality statistics that prevail in the hospital and community. This bias could be of either a demographic or clinical nature. Our objective was to define the autopsy characteristics from the National Cancer Institute of Mexico and determine how representative they were of hospital mortality. Age, sex, place of residence, socioeconomic status, and tumor location were obtained from the Hospital Mortality Registry (1985 and 1987). During these two years, 451 deaths were registered; in 290 of these cases (64.3%), an autopsy was performed. Discrepancies of 9.8 percent were found between autopsy diagnosis and mortality registry data. Our results indicate that autopsy examinations effectively reflect total hospital mortality, and represent a useful tool for epidemiological cancer studies in Mexico. Furthermore, we believe that mortality statistics should be based on autopsy results.

Pulmonary fibrogenesis after three consecutive inhalation exposures to chrysotile asbestos.

Previously, this laboratory developed a model of asbestos-induced pulmonary fibrogenesis in rats and mice after a brief (1 to 3-h) inhalation exposure. However, typical human environmental exposures would be repeated, although at lower concentrations than those used in our animal model. Here we have extended this model to encompass repeated exposures and consequent long-term effects. Groups of rats were exposed to chrysotile aerosol (10 mg/m3) for 3- to 5-h periods over 3 consecutive days. Lung fiber burden and pathologic features were studied for as long as 6 mo after exposure. We found that many of the longest (> or = 8 microm) fibers were retained in the lung for at least 6 mo, whereas shorter fibers were cleared more rapidly. The three exposures to chrysotile caused a large increase in DNA synthesis in the epithelium of terminal bronchioles and more proximal airways. When compared with a single exposure, the triple exposure caused an enhanced inflammatory response as well as a prolonged period of increased DNA synthesis in the proximal alveolar region. Hyperplastic, fibrotic lesions subsequently developed in the same region and persisted for at least 6 mo after exposure. These findings will be valuable in directing future studies of the mechanisms of pulmonary fibrosis in this model.

Ferruginous bodies as markers of environmental exposure to inorganic particles: experience with 270 autopsy cases in Mexico.

Ferruginous bodies (FB) were quantified in lung digests from 270 autopsy cases over 20 years of age. The cases were autopsied in three different hospitals of the Secretaria de Salud, Mexico, DF. Two hundred seventy samples of peripheral lung tissue were digested in commercial bleach, and all morphologic types of ferruginous bodies were quantified. The results showed that numbers of ferruginous bodies per gram of dry tissue increased over the years: 4.2 FB/g in cases from 1975 to 42.5 FB/g in cases from 1988 (r = 0.86). Higher counts of ferruginous bodies were seen in males, smokers, and Mexico City dwellers. However, more than 70% of them presented less than 100 FB/g. Our study demonstrates that most of our cases had a nonoccupational exposure to fibers.

Inorganic particles induce secretion of a macrophage homologue of platelet-derived growth factor in a density-and time-dependent manner in vitro.

The inhalation of inorganic dust can lead to the development of interstitial pulmonary fibrosis, characterized by the accumulation of fibroblasts and connective tissue matrix in the lung interstitium. The fibrosis causes alterations in the architecture of the lung parenchyma, resulting in abnormal gas exchange and hypoxemia. In a rat model of asbestos exposure, inhaled fibers are deposited on alveolar duct bifurcations, followed by an accumulation of alveolar macrophages at the sites of dust deposition. The alveolar macrophage is thought to be a major mediator of the pulmonary inflammatory response to inhaled dust. Platelet-derived growth factor (PDGF) is a cytokine that has potent chemotactic and mitogenic effects on mesenchymal cells, such as fibroblasts and smooth muscle cells. We studied the secretion of an alveolar macrophage-derived homologue of PDGF in response to carbonyl iron spheres or chrysotile asbestos fibers in vitro. We demonstrate here that rat alveolar macrophages attached to a plastic substrate produce 69 +/- 79 picograms (pg) of PDGF per 10 million macrophages. This is similar to amounts recovered from human platelets. In contrast, macrophages exposed to iron spheres secrete 429 +/- 177 pg of PDGF/10(6) macrophages after 24 h in culture. Exposure to asbestos fibers increased the PDGF production to 628 +/- 213 pg/10(6) cells. PDGF secretion was influenced by the particles in a density- and time-dependent manner. We hypothesize that PDGF and other cytokines secreted by macrophages mediate the development of dust-induced lung disease.

Differential proliferation of rat lung fibroblasts induced by the platelet-derived growth factor-AA, -AB, and -BB isoforms secreted by rat alveolar macrophages.

Platelet-derived growth factor (PDGF)-like molecules secreted by alveolar macrophages have been postulated to be mediators of lung fibrogenesis since these cytokines stimulate the proliferation and chemotaxis of lung fibroblasts. We are studying the biology and biochemistry of rat macrophage-derived PDGF and have identified for the first time the specific isoforms of PDGF (-AA, -AB, and -BB) that these macrophages secreted in vitro following activation with either chrysotile asbestos or carbonyl iron spheres. Subsequently, the proliferative response of rat lung fibroblasts (RLF) to the different PDGF isoforms was established. Using several antibodies raised against the distinct isoforms, we established that two different PDGF-like factors with molecular masses of 30 to 34 kD and 16 to 18 kD were contained in alveolar macrophage-conditioned medium. Within each of these molecular mass regions was a mixture of all three PDGF isoforms. We estimated that the 30- to 34-kD PDGF was mainly PDGF-BB (approximately 50%), while the remaining consisted of PDGF-AA (approximately 13%) and PDGF-AB (approximately 37%). Purified recombinant PDGF isoforms were tested for their ability to stimulate the growth of early-passage RLF and Swiss 3T3 cells in a 3-day cell proliferation assay. PDGF-BB and PDGF-AB were the most potent inducers of RLF proliferation and stimulated growth half-maximally at approximately 1 ng/ml and approximately 7 ng/ml, respectively. While these two B-chain-containing dimers stimulated lung fibroblast growth by as much as 150% above control medium, the PDGF-AA homodimer stimulated lung fibroblast proliferation less than 25% above control medium at the highest concentrations tested (20 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Interleukin 1 beta (IL-1 beta) and the IL-1 beta-alpha 2-macroglobulin complex upregulate the platelet-derived growth factor alpha-receptor on rat pulmonary fibroblasts.

Fibroblasts are the primary proliferating cell type in pulmonary fibrosis. We previously showed that inorganic, fibrogenic particles alter the platelet-derived growth factor (PDGF) receptor system on rat lung fibroblasts (Bonner, J. C., et al. 1993, J. Clin. Invest 92:425-430). In lung fibroblasts, PDGF is the most potent proliferative cytokine, and the responses to PDGF isoforms depend on the relative amounts of two PDGF receptors (PDGF-R alpha and PDGF-R beta). Interleukin 1 beta (IL-1 beta) production by lung macrophages is increased following exposure to fibrogenic particles. We have examined the role of IL-1 beta in regulating the lung fibroblast PDGF receptor system. IL-1 beta induced a 10-fold increase in the number of binding sites for [125I]PDGF-AA, caused a 2-fold increase in affinity of [125I]PDGF-AB, but it had no effect on [125I]PDGF-BB binding. PDGF-R alpha gene expression was increased 5-fold after 4 h of IL-1 beta treatment. IL-1 beta increased the proliferative and chemotactic response to PDGF isoforms in the following order of potency: AA > AB > BB. IL-1 beta was tested for its ability to cause increased [125I]PDGF-AA binding when complexed to its binding protein, alpha 2-macroglobulin (alpha 2M). IL-1 beta bound covalently to fast methyl-amine-activated alpha 2M (alpha 2M-MA). IL-1 beta-alpha 2M-MA or alpha 2M-MA alone possessed minimal activity for inducing an increase in [125I]PDGF-AA binding. However, treatment of the IL-1 beta-alpha 2M complex with thioredoxin, which released bioactive IL-1 beta that was covalently bound to alpha 2M, maximally increased [125I]PDGF-AA binding to the same extent as free IL-1 beta. These results indicate that the fibroblast response to PDGF isoforms is modulated by a complex interaction involving IL-1 beta, alpha 2M, and thioredoxin, all of which are produced in vivo by activated macrophages.

Transforming growth factor beta 1 downregulates the platelet-derived growth factor alpha-receptor subtype on human lung fibroblasts in vitro.

Fibroblasts are the central target cell in pulmonary fibrotic diseases, and their proliferation is mediated largely by platelet-derived growth factor (PDGF) isoforms secreted by activated lung macrophages. Several other macrophage-derived cytokines that are increased during fibrogenesis, including interleukin-1 beta and transforming growth factor-beta 1 (TGF-beta 1), could potentially modulate the mitogenic and chemotactic activity of PDGF by altering the expression of cell-surface PDGF receptors on fibroblasts. The PDGF receptor system on fibroblasts from a variety of tissues shows heterogeneous responses to TGF-beta 1. Lung fibroblasts have not been investigated in this regard. TGF-beta 1 downregulated the gene expression of the 6.5 kb PDGF-alpha receptor (PDGF-R alpha) transcript in normal human lung fibroblasts in a concentration-dependent fashion that was maximal at 3 ng/ml TGF-beta 1; this corresponded with a decrease in cell-surface PDGF-R alpha as measured by radioligand binding assays using [125I]PDGF-AA. The TGF-beta 1-induced down-regulation of the PDGF-R alpha gene was rapid (maximal suppression by 2 h post-treatment) and preceded the decrease in cell-surface alpha-receptor (maximal reduction by 6 h post-treatment). TGF-beta 1 treatment did not alter the rate of PDGF-R alpha mRNA degradation following the inhibition of transcription using actinomycin D, indicating that TGF-beta 1 increases PDGF-R alpha transcription. Scatchard analysis of saturation binding data showed that TGF-beta 1 decreased the number of [125I]PDGF-AA binding sites 5-fold without affecting receptor affinity. [125I]PDGF-AB binding sites were downregulated approximately 25%, and the number of [125I]PDGF-BB binding sites was not changed by TGF-beta 1 treatment, indicating that the PDGF-beta receptor was not affected. TGF-beta 1 reduced the mitogenic and chemotactic response to PDGF-AA by > 90%, whereas these biologic response to PDGF-AB and PDGF-BB were inhibited 50% to 80%. The proliferative and chemotactic responses of fibroblasts during tissue remodeling or during lung fibrosis are likely controlled by a complex network involving PDGF isoforms and cytokines that modify the PDGF receptor system.

Rat alveolar macrophage-derived platelet-derived growth factor is chemotactic for rat lung fibroblasts.

Alveolar macrophages and their products are thought to be important mediators of the inflammatory lesions and consequent interstitial fibrosis caused by inhalation of inorganic particles. Identification of a homolog of platelet-derived growth factor (PDGF) produced by rat alveolar macrophages that were stimulated with carbonyl iron particles and asbestos fibers motivated our studies on the biologic activity of this potent cytokine. Macrophage-derived PDGF (MD-PDGF) competes for specific membrane receptors on rat lung fibroblasts, initiating DNA synthesis and cell replication. The present report demonstrates that purified human PDGF and the MD-PDGF are chemotactic for early passage rat lung fibroblasts, but not for lung macrophages. Rat lung fibroblasts exhibit a typical bell-shaped, dose-related curve and respond optimally between 2 and 4 ng/ml PDGF. We found that alveolar macrophage-conditioned medium (AMCM), fractionated by gel filtration in 1 M acetic acid, induced a clear chemotactic response in the same fractions (20 to 22 ml) where PDGF was identified by enzyme immunoassay. In contrast, AMCM fractionated by gel filtration in phosphate-buffered saline did not induce any chemotactic activity unless the fractions were treated further with 1 M acetic acid. In this case, chemotactic activity was observed in those fractions with molecular weights of 150 and greater than 200 kD. All chemotactic activity observed with fractionated AMCM was blocked greater than 90% by an anti-PDGF antibody. These observations demonstrate that MD-PDGF is chemotactic for rat lung fibroblasts if it first is released from its binding protein, alpha-macroglobulin (alpha-M), which is secreted into the medium along with PDGF.(ABSTRACT TRUNCATED AT 250 WORDS)

PDGF-stimulated fibroblast proliferation is enhanced synergistically by receptor-recognized alpha 2-macroglobulin.

alpha-Macroglobulins derived from plasma or secreted by macrophages are platelet-derived growth factor (PDGF) binding proteins that compete with cell-surface receptors on fibroblasts for PDGF binding. alpha 2-Macroglobulin (alpha 2M) derived from bovine plasma was tested for its ability to modulate the PDGF-induced proliferation of primary passage rat lung fibroblasts (RLFs) and a human skin fibroblast cell line (CRL 1508). Fibroblasts were grown in 10% fetal bovine serum (FBS) for 24 hr, then washed with serum-free medium before adding serum-free defined medium (SFDM) containing insulin and transferrin. To this medium were added varying concentrations of human plasma-derived AB-PDGF and alpha 2 M, alone or in combination. Receptor-recognized alpha 2M was prepared by treatment with methylamine. Both native alpha 2M and the alpha 2M-methylamine (alpha 2M-MA) were tested for growth promoting activity in the absence or presence of PDGF. After 3 days, a concentration-dependent growth curve of fibroblast proliferation was demonstrated for PDGF alone, with near maximal stimulation reached at 15-20 ng/ml PDGF. alpha 2M and alpha 2M-MA alone had no effect on cell proliferation. However, alpha 2M-MA concentrations above 32 micrograms/ml synergistically enhanced PDGF-stimulated proliferation greater than 100% in the presence of 15 ng/ml PDGF. Native alpha 2M enhanced PDGF-stimulated growth 80-100% above PDGF controls only at low concentrations (32-64 micrograms/ml alpha 2M). High concentrations of native alpha 2M (128-256 micrograms/ml) either had no effect on growth or were inhibitory to PDGF-stimulated growth, depending on the cell type tested. Rat lung fibroblasts were shown to secrete a factor(s) that inhibited the trypsin-binding capacity of native alpha 2M. We further demonstrated that early passage RLFs possess specific cell-surface receptors for [125I]-PDGF and [125I]-alpha 2M-MA, and preincubation of RLFs with alpha 2M-MA increased the specific binding of [125I]-PDGF to the cell surface of these fibroblasts. Considered together, these data support the view that receptor-recognized alpha 2M synergistically enhances the proliferative capacity of PDGF. We postulate that receptor-recognized alpha Ms enhance PDGF-stimulated growth by increasing the local concentration of PDGF at the cell surface, where the PDGF could be released in close proximity to its own receptors.

Early-passage rat lung fibroblasts do not migrate in vitro to transforming growth factor-beta.

Lung fibrosis has been postulated to be mediated by the production of macrophage-derived growth factors that are both mitogenic and chemotactic for fibroblasts. In vitro studies from our laboratory demonstrated that alveolar and interstitial macrophages treated with iron and asbestos release platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) into the media. This conditioned media was capable of inducing proliferation and chemotaxis of primary rat lung fibroblasts (RLF). TGF-beta is known to be present in the media, and RLF have high-affinity receptors for TGF-beta. However, we found that > 95% of the chemotaxis was blocked by a polyclonal anti-PDGF antibody, whereas anti-TGF-beta did not change cell migration. TGF-beta has been described previously as a potent chemoattractant for fibroblasts. Thus, we tested the potential of purified TGF-beta to induce RLF chemotaxis in an attempt to address this apparent contradiction in results. Four separate preparations of RLFs from four different rats, Swiss 3T3 cells, human and rat fetal skin fibroblasts, and human foreskin fibroblasts were tested for chemotaxis using purified porcine TGF-beta 1 as well as human TGF-beta. None of these cells responded chemotactically to TGF-beta over a broad range of concentrations used (0.004 pg/ml to 50 ng/ml). RLF plated at different densities also did not respond to TGF-beta. On the other hand, all the fibroblast types migrated vigorously to PDGF (4 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Lipopolysaccharide up-regulates platelet-derived growth factor (PDGF) alpha-receptor expression in rat lung myofibroblasts and enhances response to all PDGF isoforms.

Differential expression of PDGF receptor alpha and beta subunits controls the response of mesenchymal cells to the three PDGF isoforms (AA, AB, and BB). Cultured rat lung myofibroblasts (RLMF) possess abundant PDGF receptor-beta (PDGF-Rbeta) and little PDGF receptor-alpha (PDGF-Ralpha). Here we show that LPS up-regulates expression of PDGF-Ralpha and increases the sensitivity of RLMF to all three PDGF isoforms. Treatment of RLMF for 4 to 48 h with LPS enhanced PDGF-Ralpha surface expression and mRNA 5- to 10-fold but caused no change in expression of PDGF-Rbeta. Both RNA and protein synthesis were necessary for up-regulation of PDGF-Ralpha, and the increase in PDGF-Ralpha mRNA was most likely regulated at the transcriptional level. PDGF-Ralpha up-regulation was not mediated by the IL-1R system and was independent of LPS-binding proteins in serum. Highly confluent cultures of RLMF responded more strongly to LPS than did subconfluent cultures. LPS treatment enhanced the mitogenic and chemotactic responses of RLMF to all PDGF isoforms at least threefold. We postulate that signal transduction by PDGF-receptor alphabeta heterodimers was important in the enhanced responses to PDGF-AB and -BB. We propose that regulation of PDGF-Ralpha is a critical event in the genesis of pulmonary fibroproliferative diseases.