Marjukka Myllärniemi

Inhibition of platelet-derived growth factor receptor tyrosine kinase inhibits vascular smooth muscle cell migration and proliferation.

Platelet‐derived growth factors (PDGFs) and their receptors (PDGFRs) have been linked to vascular smooth muscle cell (SMC) migration and proliferation leading to atherosclerosis, restenosis, and chronic allograft rejection. This study describes the effect of CGP 53716, a specific PDGFR tyrosine kinase inhibitor on SMC proliferation and migration in vitro and in neointimal formation in vivo. CGP 53716 inhibited dose dependently tyrosine phosphorylation of both the known PDGFRs: the PDGFR‐α and PDGFR‐β. In primary rat SMC cultures, a dose‐dependent inhibition of PDGF‐AA and PDGF‐BB induced migration, and tritiated thymidine incorporation of SMC was seen at nontoxic concentrations. After rat carotid artery ballooning injury in vivo, the migration of α‐actin‐positive cells on the luminal side of internal elastic lamina was decreased with 50 mg · kg−1 · day−1 of CGP 53716 from 38 ± 10 (control group) to 4 ± 2 (P<0.0001, Mann‐Whitney U test, N=18). CGP 53716 did not inhibit the number of replicating bromodeoxyuridine (BrdU)‐incorporating cells in the intima, media, or adventitia during BrdU labeling at 0–96 postoperative h, though it inhibited significantly (P<0.01) the replication of medial and intimal cells from 93 h onward. Intima/media ratio was inhibited by 40% after 14 days in the CGP 53716‐treated group (P=0.028) after rat aortic denudation. The results indicate that inhibition of the PDGFR tyrosine kinase inhibits SMC migration and proliferation in vitro, SMC migration, and, to a lesser extent, proliferation after ballooning injury in vivo, confirming a causal role for activation of the PDGFR and the formation of neointimal lesions.—Myllärniemi, M., Calderon, L., Lemström, K., Buchdunger, E., Häyry, P. Inhibition of platelet‐derived growth factor receptor tyrosine kinase inhibits vascular smooth muscle cell migration and proliferation. FASEB J. 11, 1119–1126 (1997)

Stabile D-peptide analog of insulin-like growth factor-1 inhibits smooth muscle cell proliferation after carotid ballooning injury in the rat.

Restenosis after angioplasty is believed to result from stimulation of smooth muscle cells (SMC) by various growth‐promoting factors as a consequence of endothelial injury. In this study we have tested the hypothesis that insulin‐like growth factor‐1 (IGF‐1)/IGF‐1 receptor (IGF‐1R) interaction is a rate‐limiting step for SMC replication by blocking this interaction with a synthetic D‐amino acid peptide structurally resembling the D‐domain of IGF‐1. After rat carotid artery denudation, semiquantitative PCR analysis demonstrated a sig‐nificant elevation of IGF‐1, platelet‐derived growth factor B, transforming growth factor βl, and epider‐mal growth factor mRNAs 10 days after endothelial injury, concomitantly with the induction of intimai SMC proliferation and intimai thickening. Admini‐stration of 10‐30 μg·kg‐l·day‐l of D‐analog of IGF‐1, devoid of proteolytic degradation in body fluids, reduced intimai SMC replication by 60‐70%. The peptide also inhibited [3H]TdR incorporation and [3H]glycine incorporation in cultured SMCs by 60‐80%, whereas a “scrambled” control peptide consisting of the same amino acids had no effect. The results suggest that IGF‐ 1/IGF‐ 1R interaction is a rate‐limiting step for SMC replication. Blocking of this interaction with stabile D‐peptide analog of IGF‐l at the level of IGF‐1R may offer an entirely new approach for the prophylaxis and treatment of reetenoeis after cardiac revascularization procedures.—Häyry, P., Myllärniemi, M., Einari, A., Alatalo, S., Aho, P., Yilmaz, S., Räisänen‐Sokolowski, A., Cozzone, G., Jameson, B. A., Baserga, R. Stable D‐peptide analog of insulin‐like growth factor‐1 inhibits smooth muscle cell proliferation after carotid ballooning injury in the rat. FASEB J. 9, 1336‐1344 (1995)

Mycophenolate mofetil (MMF, RS-61443) inhibits inflammation and smooth muscle cell proliferation in rat aortic allografts

To investigate the impact of mycophenolate mofetil (MMF) on allograft arteriosclerosis (chronic rejection) in rat aortic allograft model, we administrated MMF 20 mg/kg/day from the day of transplantation and sacrificed the rats at 1-12 months afterwards. MMF significantly suppressed all major histological manifestations of allograft arteriosclerosis, i.e. adventitial inflammation, media necrosis and intimal thickening and cellularity. There was a significant decrease in the replication rate (3H-thymidine incorporation) of inflammatory cells in the adventitia and of smooth muscle cells (SMC) in the media. MMF did not have any major effect on mRNA expression of several growth factors, (determined by polymerase chain reaction with inbuilt glyceraldehyde-3-phosphate dehydrogenase control), which have previously been demonstrated to be elevated in nonimmunosuppressed allografts. Immunoperoxidase staining showed a 40% reduction in the number of adventitial interleukin-2 receptors expressing lymphoid cells in MMF-treated allografts. The intensity of SMC alpha-actin staining was also significantly reduced. As the results suggested that MMF may have a direct antiproliferative effect on SMC, this possibility was investigated in primary SMC cultures in vitro and using the carotid denudation model in vivo. Both approaches showed inhibition of SMC proliferation by MMF. Our results indicate that MMF inhibits histopathological changes of chronic rejection by reducing the immune response and possible replication of SMC.

Selective tyrosine kinase inhibitor for the platelet-derived growth factor receptor in vitro inhibits smooth muscle cell proliferation after reinjury of arterial intima in vivo.

Summary. The long-term success of coronary angioplasty is limited by restonosis. This study was undertaken to investigate whether and to what extent the enhanced proliferative response observed in a balloon reinjury model of rat aorta is regulated by the PDGF receptor (PDGF-R). Balloon injury was performed to 14-day-old pre-existing neointimal lesion in rat aorta. PDGF receptor and ligand immunoreactivity were measured at several time points after the first and second injury, and PDGF-R signaling was blocked with a selective inhibitor of PDGF-R tyrosine kinase. In the neointima, after repeated injury, upregulation of PDGF-AA was seen to coincide with a prompt proliferative response of smooth muscle cells (SMC). Administration of the PDGF-R tyrosine kinase inhibitor in vivo, tested and found to inhibit the proliferation of SMC induced by PDGF-AA and PDGF-BB, but not by IGF-1, EGF, or bFGF, resulted in a 60% reduction in the absolute number and percentage of BrdU+ cells after the second balloon injury to pre-existing neointima, but had no significant effect on proliferation after the first injury. Endpoint lesion are was reduced by 50% in the treated group at 14 days after the second injury. The results suggest that systemic administration of a tyrosine kinase inhibitor specific for the PDGF-R can be useful in the prevention of restenosis.

Increased oxidative stress in asymptomatic current chronic smokers and GOLD stage 0 COPD

BackgroundChronic obstructive pulmonary disease (COPD) is associated with increased oxidative and nitrosative stress. The aim of our study was to assess the importance of these factors in the airways of healthy smokers and symptomatic smokers without airway obstruction, i.e. individuals with GOLD stage 0 COPD.MethodsExhaled NO (FENO) and induced sputum samples were collected from 22 current smokers (13 healthy smokers without any respiratory symptoms and 9 with symptoms i.e. stage 0 COPD) and 22 healthy age-matched non-smokers (11 never smokers and 11 ex-smokers). Sputum cell differential counts, and expressions of inducible nitric oxide synthase (iNOS), myeloperoxidase (MPO), nitrotyrosine and 4-hydroxy-2-nonenal (4-HNE) were analysed from cytospins by immunocytochemistry. Eosinophil cationic protein (ECP) and lactoferrin were measured from sputum supernatants by ELISA.ResultsFENO was significantly decreased in smokers, mean (SD) 11.0 (6.7) ppb, compared to non-smokers, 22.9 (10.0), p < 0.0001. Induced sputum showed increased levels of neutrophils (p = 0.01) and elevated numbers of iNOS (p = 0.004), MPO (p = 0.003), nitrotyrosine (p = 0.003), and 4-HNE (p = 0.03) positive cells in smokers when compared to non-smokers. Sputum lactoferrin levels were also higher in smokers than in non-smokers (p = 0.02). Furthermore, we noted four negative correlations between FENO and 1) total neutrophils (r = -0.367, p = 0.02), 2) positive cells for iNOS (r = -0.503, p = 0.005), 3) MPO (r = -0.547, p = 0.008), and 4) nitrotyrosine (r = -0.424, p = 0.03). However, no major differences were found between never smokers and ex-smokers or between healthy smokers and stage 0 COPD patients.ConclusionOur results clearly indicate that several markers of oxidative/nitrosative stress are increased in current cigarette smokers compared to non-smokers and no major differences can be observed in these biomarkers between non-symptomatic smokers and subjects with GOLD stage 0 COPD.

Prevention of Cardiac Allograft Arteriosclerosis by Protein Tyrosine Kinase Inhibitor Selective for Platelet-Derived Growth Factor Receptor

BACKGROUND Increased immunoreactivity of platelet-derived growth factor (PDGF)-AA, -Ralpha, and -Rbeta in intimal cells correlates with the development of cardiac allograft arteriosclerosis, a condition for which there is little or no current therapy. Therefore, we hypothesized that PDGF may have a rate-limiting role in the development of this disease. METHODS AND RESULTS The hypothesis was tested in a rat model of heterotopic cardiac and aortic allografts using dark agouti (AG-B4, RT1(a)) donors and Wistar-Furth (AG-B2, RT1(u)) recipients. The recipients received CGP 53716, a selective PDGF-R protein tyrosine kinase inhibitor, 50 mg. kg-1. d-1, or vehicle for 60 days. Cardiac allograft recipients also received background cyclosporin A immunosuppression. Our results demonstrate that CGP 53716 significantly reduced the incidence and intensity of arteriosclerotic lesions in rat cardiac and aortic allograft recipients. When rat coronary smooth muscle cells were stimulated in vitro with PDGF-AA or -BB in the presence of interleukin-1beta or tumor necrosis factor-alpha, CGP 53716 significantly inhibited only AA-ligand-induced but not BB-ligand-induced replication. Concomitantly, in quantitative reverse transcriptase-polymerase chain reaction, interleukin-1beta or tumor necrosis factor-alpha stimulation specifically upregulated the expression of PDGF-Ralpha mRNA but not of other ligand or receptor genes in cultured smooth muscle cells. CONCLUSIONS We conclude that a PDGF-AA/Ralpha-dependent cycle is induced in the generation of allograft arteriosclerosis that may be inhibited by blocking of signaling downstream of PDGF-R.

Proteomics of human lung tissue identifies surfactant protein A as a marker of chronic obstructive pulmonary disease.

Chronic Obstructive Pulmonary Disease (COPD), a lung disease related to smoking, is one of the leading causes of chronic morbidity and mortality around the world. One goal in COPD research is the identification of biomarkers for early diagnosis of the disease. Here, we sought COPD-specific changes in the proteome from human lung tissue. This revealed increased levels of surfactant protein A (SP-A) in COPD but not in the normal or fibrotic lung. The results were confirmed by immunohistochemistry, morphometry and Western blotting. Furthermore, elevated SP-A protein levels were detected from the induced sputum supernatants of COPD patients. The levels of other surfactant proteins (SP-B, SP-C, SP-D) were not altered. Our results suggest that SP-A is linked to the pathogenesis of COPD and could be considered as a potential COPD biomarker.

8-Isoprostane as a marker of oxidative stress in nonsymptomatic cigarette smokers and COPD

8-Isoprostane is a potential in vivo marker for oxidant burden, but its usefulness in induced sputum of smokers and chronic obstructive pulmonary disease (COPD) has not been investigated. The current study investigated 58 subjects comprising 11 never-smokers, 11 ex-smokers, 13 healthy current smokers and 23 COPD with stage 0–III disease (according to the Global Initiative for Chronic Obstructive Lung Disease criteria). 8-Isoprostane was determined from induced sputum by enzyme immunoassay. Sputum 8-isoprostane levels were similar in the never-smokers and ex-smokers, but were elevated in the healthy smokers compared with nonsmokers, and in those with stage I–III COPD. Sputum 8-isoprostane levels could not differentiate nonsymptomatic smokers from those with Stage 0 COPD. There was a correlation between sputum 8-isoprostane level and lung function parameters (forced expiratory volume in one second/forced vital capacity and sputum neutrophils. In conclusion, sputum 8-isoprostane levels correlate with the severity of chronic obstructive pulmonary disease. However, they do not appear to differentiate healthy smokers from those who are at risk of developing chronic obstructive pulmonary disease (Global Initiative for Chronic Obstructive Lung Disease stage 0).

Airway biomarkers of the oxidant burden in asthma and chronic obstructive pulmonary disease: Current and future perspectives

The pathogenesis of asthma and chronic obstructive pulmonary disease (COPD) has been claimed to be attributable to increased systemic and local oxidative stress. Detection of the oxidant burden and evaluation of their progression and phenotypes by oxidant biomarkers have proved challenging and difficult. A large number of asthmatics are cigarette smokers and smoke itself contains oxidants complicating further the use of oxidant biomarkers. One of the most widely used oxidant markers in asthma is exhaled nitric oxide (NO), which plays an important role in the pathogenesis of asthma and disease monitoring. Another oxidant marker that has been widely investigated in COPD is 8-isoprostane, but it is probably not capable of differentiating asthma from COPD, or even sensitive in the early assessment of these diseases. None of the current biomarkers have been shown to be better than exhaled NO in asthma. There is a need to identify new biomarkers for obstructive airway diseases, especially their differential diagnosis. A comprehensive evaluation of oxidant markers and their combinations will be presented in this review. In brief, it seems that additional analyses utilizing powerful tools such as genomics, metabolomics, lipidomics, and proteomics will be required to improve the specificity and sensitivity of the next generation of biomarkers.

Oxidant–Antioxidant Imbalance as a Potential Contributor to the Progression of Human Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is the most common idiopathic interstitial pneumonia. IPF is a disease with poor prognosis and an aggressive nature, and poses major challenges to clinicians. Thus, a large part of research in the area has focused on the pathogenesis on IPF. Characteristic features in IPF include fibrotic lesions devoid of inflammatory cell infiltrates. There are experimental models of lung fibrosis (e.g., bleomycin-induced fibrosis), but they typically contain a prominent inflammatory pattern in the lung, which leads to relatively diffuse lung fibrosis. Nonetheless, experimental models have provided important information about the progression and pathways contributing to the lung fibrosis, including activation of transforming growth factor beta (TGF-beta). Both patient material and experimental models of lung fibrosis have displayed marked elevation of several markers of oxidant burden and signs for disturbed antioxidant/oxidant balance. Several studies also suggest that reactive oxygen species can cause activation of growth-regulatory cytokines, including TGF-beta. In addition, there are indications that endogenous and exogenous antioxidants/redox modulators can influence fibrogenesis, protect the lung against fibrosis, and prevent its progression. Factors that restore the antioxidant capacity and prevent sustained activation of growth-regulatory cytokines may have a therapeutic role in IPF.