Carbon ion radiotherapy of human lung cancer attenuates HIF-1 signaling and acts with considerably enhanced therapeutic efficiency.

Carbon ion irradiation is an emerging therapeutic option for various tumor entities. Radiation resistance of solid tumors toward photon irradiation is caused by attenuation of DNA damage in less oxygenated tumor areas and by increased hypoxia-inducible factor (HIF)-1 signaling. Carbon ion irradiation acts independently of oxygen; however, the role of HIF-1 is unclear. We analyzed the effect of HIF-1 signaling after carbon ions in comparison to photons by using biological equivalent radiation doses in a human non-small-cell cancer model. The studies were performed in cultured A549 and H1299 cell lines and in A549 xenografts. Knockdown of HIF-1α in vivo combined with photon irradiation delayed tumor growth (23 vs. 13 d; P<0.05). Photon irradiation induced HIF-1α and target genes, predominantly in oxygenated cells (1.6-fold; P<0.05), with subsequent enhanced tumor angiogenesis (1.7-fold; P<0.05). These effects were not observed after carbon ion irradiation. Micro-DNA array analysis indicated that photons, but not carbon ions, significantly induced components of the mTOR (mammalian target of rapamycin) pathway (gene set enrichment analysis; P<0.01) as relevant for HIF-1α induction. After carbon ion irradiation in vivo, we observed substantially decreased HIF-1α levels (8.9-fold; P<0.01) and drastically delayed tumor growth (P<0.01), an important finding that indicates a higher relative biological effectiveness (RBE) than anticipated from the cell survival data. Taken together, the evidence showed that carbon ions mediate an improved therapeutic effectiveness without tumor-promoting HIF-1 signaling.

Inhibition of matrix deposition: a new strategy for prevention of restenosis after balloon angioplasty.

Restenosis after balloon angioplasty or stent placement is characterized by local accumulation of mainly vascular smooth muscle cells and the synthesis of extracellular matrix molecules (ECM).We hypothesized that inhibition of ECM synthesis represents a strategy to prevent trauma-induced neointima formation. Rats were treated with pirfenidone (1 g/kg of body weight orally.), an inhibitor of growth factor-induced collagen synthesis, and subjected to balloon denudation of the carotid artery. Two weeks later, computer-aided morphometry was done and compared with untreated controls (each n = 6). Neointimal proliferative activity was quantified immunohistochemically by counting PCNA-positive nuclei, and collagen deposition was visualized by picrosirius red staining and semi-quantified by Northern blot. Control-injured animals developed marked neointimal thickening within 2 weeks (I/M, mean intima to media ratio: 2.42 +/- 0.15) resulting in an 89.2% luminal narrowing. The neointima mainly consisted of vascular smooth muscle cells embedded in collagen. Neointima formation was strongly reduced when balloon-injured animals had been treated with pirfenidone (I/M ratio 0.22 +/- 0.08, P < 0.001), resulting in a minimal residual narrowing of the lumen (7.9%). I/M ratio did not further increase even after discontinuation of the drug for 14 days (0.35 +/- 0.13). Proliferative activity within the neointima was unaffected by the drug, 4.4% versus 4.8% of neointimal cells stained positive for PCNA in carotid arteries of treated versus untreated animals, respectively. However, picrosirius red staining demonstrated marked attenuation of collagen deposition, a finding that was further confirmed by Northern blot of homogenized vessels. Pirfenidone, currently being investigated clinically for the treatment of various fibrotic diseases, is able to prevent neointimal lesion formation most likely through inhibition of local ECM deposition. Targeting matrix deposition may have an intriguing potential for the prevention of vascular proliferative diseases.

Calpain counteracts mechanosensitive apoptosis of vascular smooth muscle cells in vitro and in vivo.

Mechanical forces contribute to vascular remodeling processes. Elevated mechanical stress causes apoptosis of vascular smooth muscle cells (VSMCs) within the media. This study examined the role of the cystein protease calpain in force-induced vascular cell apoptosis and its effect on injury-induced vascular remodeling processes. VSMCs were exposed to cyclic tensile force in vitro, which resulted in increased p53 protein expression and transcriptional activity as well as a significant increase of apoptotic VSMCs. Apoptosis was prevented by the p53 inhibitor pifithrin and by p53 antisense oligonucleotides, indicating dependency of force-induced apoptosis on p53. Simultaneously, calpain activity increased by mechanical stress. Prevention of calpain activation by calpeptin or antisense oligonucleotides augmented strain-induced p53 expression and transcriptional activity, resulting in a further increase of apoptotic rate. p53 protein was directly disintegrated by activated calpain. The in vivo relevance of the findings was tested: pharmacologic inhibition of initial calpain activation augmented early apoptosis of medial VSMCs 24 h after balloon injury in a p53-dependent manner but resulted in a marked increase in late neointima formation. We conclude that calpain counteracts mechanically induced excessive VSMC apoptosis through its p53-degrading properties, which identifies calpain as a key regulator of mechanosensitive remodeling processes of the vascular wall.

Spatiotemporal expression of flk-1 in pulmonary epithelial cells during lung development.

Vascular endothelial growth factor-A (VEGF-A) responsive effects mediated via the receptors fetal liver kinase-1 (flk-1) and fms-like tyrosine kinase (flt-1), are key processes of pulmonary vascular development. Flk-1 has been shown to be involved in early embryonic lung epithelial to endothelial crosstalk and branching morphogenesis. Recent reports suggested a role of VEGF-A in lung epithelial cell function. Based on these observations, we hypothesize that epithelial flk-1 has a unique function in pulmonary development. Thus, the aim of this study is to elucidate spatiotemporal expression of flk-1 during lung development with respect to the epithelial system. Embryonic lungs were screened for flk-1 messenger RNA and protein at daily intervals, including postnatal stages. From Embryonic Day (ED) 12.5 through ED 15.5, flk-1 expression was restricted to the early vascular primitive network, while from ED 16.5 on flk-1 was detectable in the epithelial system and persisted there postnatally. At postnatal stages, flk-1 expression was increasingly restricted to individual cells in the alveolar septa. Isolation and in vitro cultivation of alveolar epithelial cells confirmed flk-1 expression and showed VEGF secretion into the supernatant. To our knowledge, this is the first murine study characterizing epithelial flk-1 expression at different stages throughout lung organogenesis until birth and at postnatal stages. To confirm epithelial flk-1 expression, we performed reporter gene analysis of the flk-1 promoter in vivo. Investigations on transgenic mouse strains, containing either a complete or incomplete flk-1 promoter driving expression of the lacZ reporter gene, suggested differential flk-1 regulation in endothelial and epithelial cells.

Caveolin-1 facilitates mechanosensitive protein kinase B (Akt) signaling in vitro and in vivo.

Mechanotransduction represents an integral part of vascular homeostasis and contributes to vascular lesion formation. Previously, we demonstrated a mechanosensitive activation of phosphoinositide 3-kinase (PI3-K)/protein kinase B (Akt) resulting in p27Kip1 transcriptional downregulation and cell cycle entry of vascular smooth muscle cells (VSMC). In this study, we further elucidated the signaling from outside-in toward PI3-K/Akt in vitro and in an in vivo model of elevated tensile force. When VSMC were subjected to cyclic stretch (0.5 Hz at 125% resting length), PI3-K, Akt, and Src kinases were found activated. Disrupting caveolar structures with beta-cyclodextrin or transfection of VSMC with caveolin-1 antisense oligonucleotides (ODN) prevented PI3-K and Akt activation and cell cycle entry. Furthermore, PI3-K and Akt were resistant to activation when Src kinases were inhibited pharmacologically or by overexpression of a kinase-dead c-Src mutant. alpha(V)beta3 integrins were identified to colocalize with PI3-K/caveolin-1 complexes, and blockade of alpha(V)beta3 integrins prevented Akt activation. The central role of caveolin-1 in mechanotransduction was further examined in an in vivo model of elevated tensile force. Interposition of wild-type (WT) jugular veins into WT carotid arteries resulted in a rapid Akt activation within the veins that was almost abolished when veins of caveolin-1 knockout (KO) mice were used. Furthermore, late neointima formation within the KO veins was significantly reduced. Our study provides evidence that PI3-K/Akt is critically involved in mechanotransduction of VSMC in vitro and within the vasculature in vivo. Furthermore, caveolin-1 is essential for the integrin-mediated activation of PI3-K/Akt.

Hypoxia-driven proliferation of human pulmonary artery fibroblasts: cross-talk between HIF-1alpha and an autocrine angiotensin system.

Pulmonary artery adventitial fibroblasts (FBPA) may play a central role in lung vascular remodeling under conditions of hypoxia and inflammation, the result being pulmonary hypertension and cor pulmonale. In cultured human FBPA, both angiotensin II (Ang II) and hypoxia promoted cell cycle progression and cell proliferation and suppressed apoptosis. These effects were further enhanced when both stimuli were applied simultaneously. Hypoxia elevated the expression of hypoxia-inducible factor 1alpha (HIF-1alpha) and increased the expression of genes regulated by the hypoxia-responsive element (HRE). Up-regulation of both angiotensin-converting enzyme (ACE) and Ang II receptor type 1 (AT1) was also observed. Exogenous Ang II further increased HIF/HRE-dependent signaling in FBPA, whereas suppression of the autocrine ACE-Ang II-AT1 loop with inhibitors of ACE, AT1, and phosphatidylinositol 3-kinase (PI3K) reduced the proliferative response to both hypoxia and exogenous Ang II. Overexpression of HIF-1alpha by transient transfection caused the same proliferative effect and up-regulation of AT1 expression that were observed under hypoxic conditions. In contrast, small interfering RNA targeting HIF-1alpha inhibited hypoxia-induced ACE and AT1 expression. Our studies indicate that the ACE-Ang II-AT1 system serves as a positive feedback loop and fosters FBPA proliferation under hypoxic conditions, with the PI3K-HIF-HRE axis as the central effector pathway. This pathway may thus facilitate vascular remodeling under hypoxic conditions.

Mechanosensitive p27Kip1 regulation and cell cycle entry in vascular smooth muscle cells.

BACKGROUND: Cyclic stretch plays an important role in the homeostasis of vessel structure. Increased forces might, however, contribute to remodeling processes, resulting in vascular proliferative diseases. The initial molecular events necessary for mechanosensitive cell cycle entry of quiescent smooth muscle cells are poorly understood. METHODS AND RESULTS: In this study, we demonstrate that mechanical strain resulted in a rapid, integrin-dependent but mitogen-independent activation of phosphoinositide 3-kinase (PI3-K)/protein kinase B (Akt) in quiescent vascular smooth muscle cells. Subsequently, downstream ALL 1 fused gene from chromosome X (AFX)-like forkhead transcription factors were inactivated, leading to transcriptional downregulation of p27Kip1. This contrasted with the posttranscriptional protein reduction of p27Kip1 in cells stimulated with serum mitogens. Stretch-mediated p27Kip1 downregulation was accompanied by activation of cyclin-dependent kinase 2, hyperphosphorylation of retinoblastoma protein, and proliferation. Forkhead transcription factor inactivation and p27Kip1 downregulation were prevented by the PI3-K inhibitors wortmannin and LY294002. Pharmacological blockade of other kinases, such as p42/44, p38, and protein kinase A or C, did not influence the mechanosensitive gene regulation. p27Kip1 downregulation and cell cycle entry were, however, prevented by overexpression of a constitutively inactive form of Akt or constitutively active forms of forkhead transcription factors. CONCLUSIONS: Our data demonstrate that the earliest cell cycle events can occur in a solely mechanosensitive fashion. Vascular smooth muscle cells are, furthermore, able to use transcriptional or posttranscriptional mechanisms to regulate p27Kip1, depending on the stimulus to which they are exposed. This observation has novel implications for understanding of vascular proliferative diseases.

Information on new drugs at market entry: retrospective analysis of health technology assessment reports versus regulatory reports, journal publications, and registry reports.

BACKGROUND: When a new drug becomes available, patients and doctors require information on its benefits and harms. In 2011, Germany introduced the early benefit assessment of new drugs through the act on the reform of the market for medicinal products (AMNOG). At market entry, the pharmaceutical company responsible must submit a standardised dossier containing all available evidence of the drug's added benefit over an appropriate comparator treatment. The added benefit is mainly determined using patient relevant outcomes. The "dossier assessment" is generally performed by the Institute for Quality and Efficiency in Health Care (IQWiG) and then published online. It contains all relevant study information, including data from unpublished clinical study reports contained in the dossiers. The dossier assessment refers to the patient population for which the new drug is approved according to the summary of product characteristics. This patient population may comprise either the total populations investigated in the studies submitted to regulatory authorities in the drug approval process, or the specific subpopulations defined in the summary of product characteristics ("approved subpopulations"). OBJECTIVE: To determine the information gain from AMNOG documents compared with non-AMNOG documents for methods and results of studies available at market entry of new drugs. AMNOG documents comprise dossier assessments done by IQWiG and publicly available modules of company dossiers; non-AMNOG documents comprise conventional, publicly available sources-that is, European public assessment reports, journal publications, and registry reports. The analysis focused on the approved patient populations. DESIGN: Retrospective analysis. DATA SOURCES: All dossier assessments conducted by IQWiG between 1 January 2011 and 28 February 2013 in which the dossiers contained suitable studies allowing for a full early benefit assessment. We also considered all European public assessment reports, journal publications, and registry reports referring to these studies and included in the dossiers. DATA ANALYSIS: We assessed reporting quality for each study and each available document for eight methods and 11 results items (three baseline characteristics and eight patient relevant outcomes), and dichotomised them as "completely reported" or "incompletely reported (including items not reported at all)." For each document type we calculated the proportion of items with complete reporting for methods and results, for each item and overall, and compared the findings.Results 15 out of 27 dossiers were eligible for inclusion and contained 22 studies. The 15 dossier assessments contained 28 individual assessments of 15 total study populations and 13 approved subpopulations. European public assessment reports were available for all drugs. Journal publications were available for 14 out of 15 drugs and 21 out of 22 studies. A registry report in ClinicalTrials.gov was available for all drugs and studies; however, only 11 contained results. In the analysis of total study populations, the AMNOG documents reached the highest grade of completeness, with about 90% of methods and results items completely reported. In non-AMNOG documents, the rate was 75% for methods and 52% for results items; journal publications achieved the best rates, followed by European public assessment reports and registry reports. The analysis of approved subpopulations showed poorer complete reporting of results items, particularly in non-AMNOG documents (non-AMNOG versus AMNOG: 11% v 71% for overall results items and 5% v 70% for patient relevant outcomes). The main limitation of our analysis is the small sample size. CONCLUSION: Conventional, publicly available sources provide insufficient information on new drugs, especially on patient relevant outcomes in approved subpopulations. This type of information is largely available in AMNOG documents, albeit only partly in English. The AMNOG approach could be used internationally to develop a comprehensive publication model for clinical studies and thus represents a key open access measure.

Protective effect of 3-deazaadenosine in a rat model of lipopolysaccharide-induced myocardial dysfunction.

Severe sepsis is accompanied by a profound depression of myocardial contractility. Leukocyte adhesion with subsequent local excess nitric oxide and reactive oxygen species production play major roles for this deleterious effect. We hypothesized that 3-deazaadenosine (c3Ado), an adenosine analogue with anti-inflammatory properties, prevents endotoxin-induced myocardial dysfunction. Wistar rats (8 per group) were treated with Escherichia coli lipopoly-saccharide (LPS, 1 mg/kg, i.p., strain 0111:B4) +/- c3Ado (10 mg/kg, i.p.) 8 h before their hearts were harvested for isolated perfusion, histochemical analysis, or electrophoretic mobility shift assay. LPS induced a marked depression of left ventricular contractility. Immunohistochemistry revealed an upregulation of the adhesion molecules VCAM-1, ICAM-1, and P-selectin within the postcapillary venules. c3Ado inhibited VCAM-1 and ICAM-1 upregulation, but not P-selectin, and prevented cardiodepression. Electrophoretic mobility shift assay revealed inactivation of the transcription factor nuclear factor-kappaB and immunohistochemical staining for gp91phox, ED1, and CD11b demonstrated that c3Ado prevented local recruitment of monocytes and polymorph nuclear neutrophils to the myocardium. Accordingly, significantly fewer leukocytes producing nitric oxide or reactive oxygen species accumulated within the myocardium. Intravital microscopy of intestinal venules confirmed that LPS-induced adhesion of leukocytes was prevented by c3Ado. Additionally, c3Ado prevented LPS-induced elevation of serum tumor necrosis factor-alpha levels. Our results imply that c3Ado may prove to have clinical relevance for inflammatory disease processes.

Comparison of the effects of carbon ion and photon irradiation on the angiogenic response in human lung adenocarcinoma cells.

PURPOSE: Radiotherapy resistance is a commonly encountered problem in cancer treatment. In this regard, stabilization of endothelial cells and release of angiogenic factors by cancer cells contribute to this problem. In this study, we used human lung adenocarcinoma (A549) cells to compare the effects of carbon ion and X-ray irradiation on the cells' angiogenic response. METHODS AND MATERIALS: A549 cells were irradiated with biologically equivalent doses for cell survival of either carbon ions (linear energy transfer, 170 keV/µm; energy of 9.8 MeV/u on target) or X-rays and injected with basement membrane matrix into BALB/c nu/nu mice to generate a plug, allowing quantification of angiogenesis by blood vessel enumeration. The expression of angiogenic factors (VEGF, PlGF, SDF-1, and SCF) was assessed at the mRNA and secreted protein levels by using real-time reverse transcription-PCR and enzyme-linked immunosorbent assay. Signal transduction mediated by stem cell factor (SCF) was assessed by phosphorylation of its receptor c-Kit. For inhibition of SCF/c-Kit signaling, a specific SCF/c-Kit inhibitor (ISCK03) was used. RESULTS: Irradiation of A549 cells with X-rays (6 Gy) but not carbon ions (2 Gy) resulted in a significant increase in blood vessel density (control, 20.71 ± 1.55; X-ray, 36.44 ± 3.44; carbon ion, 16.33 ± 1.03; number per microscopic field). Concordantly, irradiation with X-rays but not with carbon ions increased the expression of SCF and subsequently caused phosphorylation of c-Kit in endothelial cells. ISCK03 treatment of A549 cells irradiated with X-rays (6 Gy) resulted in a significant decrease in blood vessel density (X-ray, 36.44 ± 3.44; X-ray and ISCK03, 4.33 ± 0.71; number of microscopic field). These data indicate that irradiation of A549 cells with X-rays but not with carbon ions promotes angiogenesis. CONCLUSIONS: The present study provides evidence that SCF is an X-ray-induced mediator of angiogenesis in A549 cells, a phenomenon that could not be observed with carbon ion irradiation. Thus, in this model system evaluating angiogenesis, carbon ion irradiation may have a therapeutic advantage. This observation should be confirmed in orthotopic lung tumor models.

Transforming growth factor-beta-dependent growth inhibition in primary vascular smooth muscle cells is p38-dependent.

Vascular smooth muscle cells (VSMCs) constitute the major cellular component of the vessel tunica media. VSMC proliferation is a key feature in developing vessels and pathological states such as atherosclerosis and restenosis. Transforming growth factor (TGF)-beta is a key regulator of VSMCs, but its effect on VSMC proliferation and apoptosis are controversial. Here, we characterized TGF-beta effects on basal-, serum-, and platelet-derived growth factor-BB-induced primary mouse VSMC proliferation. TGF-beta led to potent growth inhibition of VSMCs isolated from normal mouse aortae without inducing apoptosis. Growth inhibition by TGF-beta was due to G0/G1 arrest. Next, we explored distinct signaling pathways activated by TGF-beta and the effects of pharmacological inhibition of these. TGF-beta led to activation of Smad2/3, p38, p42/44, and c-Jun NH2-terminal kinase (JNK) pathways, assessed by phosphorylation, immunofluorescence, and reporter gene analysis. TGF-beta-dependent growth inhibition was specifically attenuated by pharmacological blockade of the TGF-beta type I receptor (TbetaRI) kinase or p38 mitogen-activated protein kinase pathways, whereas blockade of p42/44 or JNK kinases did not influence the effect of TGF-beta. TbetaRI kinase inhibition blocked all downstream pathways including Smad and p38 phosphorylation. In contrast, p38 inhibition did not alter Smad function, as assessed by translocation or reporter gene expression, but selectively inhibited p38 activity. These results demonstrate that TGF-beta acts as a potent antiproliferative mediator in VSMCs, irrespective of the proliferative stimulus, without inducing apoptotic effects. The anti-proliferative effect of TGF-beta is due to G0/G1 arrest and mediated primarily by the p38 pathway, suggesting that p38 kinase is central to TGF-beta-mediated growth inhibition in primary mouse VSMCs.