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Thomas Krieg

Publications and source records attributed to Thomas Krieg.

At least 73 records · Page 4Linked to original sources
PubMed2004

Keloids--clinical diagnosis, pathogenesis, and treatment options.

Keloids are defined as excessive scar tissue formation extending beyond the area of the original skin injury and occurring in predisposed individuals. They are considered to be a result of abnormal wound healing. The pathogenetic mechanisms that cause keloids remain unknown. Experiments with cells derived from keloid tissue revealed a number of abnormalities in cellular functions, such as in proliferation, apoptosis, or expression of growth factors and extracellular matrix proteins. Furthermore, several studies have reported altered keratinocyte-fibroblast interactions in keloids. Despite the diverse pathological changes in cellular functions and expression profiles of cells derived from keloid tissue, recent genetic studies have provided evidence that single genes may act as major regulators of keloid formation. We provide an overview of the pathogenetic mechanisms of keloid formation in the context of their clinical characteristics and current therapeutic approaches.

Dermatologic Agents↗
PubMed2004

High plasminogen activator inhibitor type 2 expression is a hallmark of scleroderma fibroblasts in vitro.

Systemic scleroderma is a chronic disease, which leads to fibrosis of the skin and internal organs. Fibroblasts obtained from patients with this disease demonstrate an activated state in culture. We, in this study, report strong, constitutive overexpression of plasminogen activator inhibitor type-2 (PAI-2) in scleroderma fibroblasts and demonstrate that this induction observed at the mRNA and protein level is dependent on serum addition. Induced PAI-2 protein levels were restricted to the non-glycosylated 47-kDa form, which is located intracellularly. Induction was stable for at least 12 passages. No modulation by fibrogenic cytokines--for example, transforming growth factor-beta1 or connective tissue growth factor--or by antagonizing IL-1 receptors was observed. The data indicate that scleroderma fibroblasts are more sensitive to the induction of PAI-2 expression than control fibroblasts by a presently unknown factor in serum.

Adult↗
PubMed2004

Genetically modified mice as a tool to study inflammatory skin diseases.

Although mice have a long tradition as models for human skin diseases, they have recently received increasingly more attention. This is because of the rapid advancement of genetic engineering methods which made it possible to create mice with precisely defined genetic changes. Many of these mice develop impressive and sometimes unexpected, puzzling phenotypes. Their interpretation is a major challenge to the basic researcher and can often be ameliorated by the input of an experienced dermatologist. Together with recent examples of genetically modified mouse models of inflammatory skin diseases we give a short overview of the methods used to generate such mice, describe possible ways to analyse them, and discuss problems that arise in the interpretation of the findings.

Animals↗
PubMed2004

Dissecting the roles of endothelin, TGF-beta and GM-CSF on myofibroblast differentiation by keratinocytes.

Myofibroblasts are specialized fibroblasts that contribute to wound healing by producing extracellular matrix and by contracting the granulation tissue. They appear in a phase of wound healing when the dermis strongly interacts with activated epidermal keratinocytes. Direct co-culture with keratinocytes upregulates TGFbeta activity and also induces fibroblast to differentiate into alpha-smooth muscle actin (alphaSMA)-positive myofibroblasts. TGF-beta activity alone cannot completely account for alphaSMA induction in these co-cultures, and here we analyze mechanical force generation, another potent inducer of myofibroblast differentiation in this model. Using deformable silicone substrates, we show that contractile activity of fibroblasts is already induced after 1-2-days of co-culture, when fibroblasts are generally alphaSMA negative. Endothelin-1 (ET-1), the most potent inducer of smooth muscle cell contraction, was up-regulated in co-cultures, while blocking ET-1 with the ET receptor inhibitor PD156252 inhibited contraction in these early co-cultures. In 4-5 days of co-culture, however, fibroblast contractile activity correlated with an increased expression of alphaSMA expression. Stimulation of fibroblast mono-cultures with ET-1 in a low serum medium did not induce alphaSMA expression; however, ET-1 did synergize with TGF-beta. Surprisingly, GM-CSF, another mediatorstimulating myofibroblast differentiation in granulation tissue, inhibited alphaSMA expression in fibroblasts, costimulated with TGF-beta and ET-1. GM-CSF activated NFkappaB, thus interfering with TGF-beta signaling. Blocking TGFbeta and ET-1 largely impaired alphaSMA induction in co-cultures at day 7 and, in combination, almost completely prevented alphaSMA induction. Our results dissect the roles of TGF-beta and ET-1 on mechanical force generation in keratinocyte-fibroblast co-cultures, and identify GM-CSF as an inducer of myofibroblasts acting indirectly.

Antibodies, Monoclonal↗
PubMed2004

Gene transfer in tissue repair: status, challenges and future directions.

Wound repair involves a complex interaction of various cell types, extracellular matrix molecules and soluble mediators. Details on signals controlling wound cell activities are beginning to emerge. In recent years this knowledge has been applied to a number of therapeutic strategies in soft tissue repair. Key challenges include re-adjusting the adult repair process in order to augment diseased healing processes, and providing the basis for a regenerative rather than a reparative wound environment. In particular, the local delivery of pluripotent growth factor molecules to the injured tissue has been intensively investigated over the past decade. Limited success of clinical trials indicates that an important aspect of the growth factor wound-healing paradigm is the effective delivery of these polypeptides to the wound site. A molecular genetic approach in which genetically modified cells synthesise and deliver the desired growth factor in a time-regulated manner is a powerful means to overcome the limitations associated with the (topical) application of recombinant growth factor proteins. This article summarises repair mechanisms and their failure, and gives an overview of techniques and studies applied to gene transfer in tissue repair. It also provides perspectives on potential targets for gene transfer technology.

Adenoviridae↗
PubMed2003

CD4+ T cell-associated pathophysiology critically depends on CD18 gene dose effects in a murine model of psoriasis.

In a CD18 hypomorphic polygenic PL/J mouse model, the severe reduction of CD18 (beta(2) integrin) to 2-16% of wild-type levels leads to the development of a psoriasiform skin disease. In this study, we analyzed the influence of reduced CD18 gene expression on T cell function, and its contribution to the pathogenesis of this disease. Both CD4(+) and CD8(+) T cells were significantly increased in the skin of affected CD18 hypomorphic mice. But only depletion of CD4(+) T cells, and not the removal of CD8(+) T cells, resulted in a complete clearance of the psoriasiform dermatitis. This indicates a central role of CD4(+) T cells in the pathogenesis of this disorder, further supported by the detection of several Th1-like cytokines released predominantly by CD4(+) T cells. In contrast to the CD18 hypomorphic mice, CD18 null mutants of the same strain did not develop the psoriasiform dermatitis. This is in part due to a lack of T cell emigration from dermal blood vessels, as experimental allergic contact dermatitis could be induced in CD18 hypomorphic and wild-type mice, but not in CD18 null mutants. Hence, 2-16% of CD18 gene expression is obviously sufficient for T cell emigration driving the inflammatory phenotype in CD18 hypomorphic mice. Our data suggest that the pathogenic involvement of CD4(+) T cells depends on a gene dose effect with a reduced expression of the CD18 protein in PL/J mice. This murine inflammatory skin model may also have relevance for human polygenic inflammatory diseases.

Animals↗
PubMed2003

Bradykinin induces mitochondrial ROS generation via NO, cGMP, PKG, and mitoKATP channel opening and leads to cardioprotection.

Bradykinin (BK) mimics ischemic preconditioning by generating reactive oxygen species (ROS). To identify intermediate steps that lead to ROS generation, rabbit cardiomyocytes were incubated in reduced MitoTracker Red stain, which becomes fluorescent after exposure to ROS. Fluorescence intensity in treated cells was expressed as a percentage of that in paired, untreated cells. BK (500 nM) caused a 51 +/- 16% increase in ROS generation (P < 0.001). Coincubation with either the BK B2-receptor blocker HOE-140 (5 microM) or the free radical scavenger N-(2-mercaptopropionyl)glycine (1 mM) prevented this increase, which confirms that the response was receptor mediated and ROS were actually being measured. Closing mitochondrial ATP-sensitive K+ (mitoKATP) channels with 5-hydroxydecanoate (5-HD, 1 mM) prevented increased ROS generation. BK-induced ROS generation was blocked by Nomega-nitro-m-arginine methyl ester (m-NAME, 200 microM), which implicates nitric oxide as an intermediate. Blockade of guanylyl cyclase with 1-H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 10 microM) aborted BK-induced ROS generation but not that from diazoxide, a direct opener of mitoKATP channels. The protein kinase G (PKG) blocker 8-bromoguanosine-3',5'-cyclic monophosphorothioate (25 microM) eliminated the effects of BK. Conversely, direct activation of PKG with 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (100 microM) increased ROS generation (39 +/- 15%; P < 0.004) similar to BK. This increase was blocked by 5-HD. Finally, the nitric oxide donor S-nitroso-N-acetylpenicillamine (1 microM) increased ROS by 34 +/- 6%. This increase was also blocked by 5-HD. In intact rabbit hearts, BK (400 nM) decreased infarction from 30.5 +/- 3.0 of the risk zone in control hearts to 11.9 +/- 1.4% (P < 0.01). This protection was aborted by either 200 microM m-NAME or 2 microM ODQ (35.4 +/- 5.7 and 30.4 +/- 3.0% infarction, respectively; P = not significant vs. control). Hence, BK preconditions through receptor-mediated production of nitric oxide, which activates guanylyl cyclase. The resulting cGMP activates PKG, which opens mitoKATP. Subsequent release of ROS triggers cardioprotection.

Adenosine Triphosphate↗
PubMed2003

Invasion of melanoma cells into dermal connective tissue in vitro: evidence for an important role of cysteine proteases.

Invasion of melanoma cells into the dermal connective tissue is a major characteristic in the complex process of metastasis. Proteases play an important role in tumor cell invasion as these enzymes are able to degrade most components of the extracellular matrix (ECM), and thus enable cells to penetrate interstitial connective tissues and basement membranes. We developed an improved culture model that allows the detailed study of melanoma cell invasion in vitro. In this model, high (BLM) or low (530) invasive melanoma cells were seeded on the dermal side of dead deepidermized dermis (DDD) and cultured for 14 days at the air/liquid interface. The high invasive cells invaded the tissue, leading to dermal tumor formation, whereas the low invasive cells did not. Analysis of the enzymatic activity of gelatinases by in situ gelatin zymography at neutral pH revealed proteolysis only in those composites cultured with high invasive melanoma cells. Interestingly, in situ zymograms performed at more acidic conditions, favoring the activity of cysteine proteases, exhibited markedly enhanced and widespread gelatinolysis compared to neutral pH. Cysteine protease inhibitors (E-64 and leupeptin) significantly reduced invasion of melanoma cells into these composites. These results indicate an important role of cysteine proteases for tumor invasion.

Cathepsins↗
PubMed2003

Multiple integrin-ligand interactions synergize in shear-resistant platelet adhesion at sites of arterial injury in vivo.

Damage to the integrity of the vessel wall results in exposure of the subendothelial extracellular matrix (ECM), which triggers integrin-dependent adhesion and aggregation of platelets. The role of platelet beta1 integrins in these processes remains mostly undefined. Here, we demonstrate by intravital fluorescence microscopy that platelet adhesion and thrombus growth on the exposed ECM of the injured carotid artery is not significantly altered in alpha2-null mice and even in mice with a Cre/loxP-mediated loss of all beta1 integrins on their platelets. In contrast, inhibition of alphaIIbbeta3 integrin on platelets in wild-type mice blocked aggregate formation and reduced platelet adhesion by 60.0%. Strikingly, alphaIIbbeta3 inhibition had a comparable effect in alpha2-null mice, demonstrating that other receptors mediate shear-resistant adhesion in the absence of functional alpha2beta1 and alphaIIbbeta3. These were identified to be alpha5beta1 and/or alpha6beta1 as alphaIIbbeta3 inhibition abrogated platelet adhesion in beta1-null mice. We conclude that shear-resistant platelet adhesion on the injured vessel wall in vivo is a highly integrated process involving multiple integrin-ligand interactions, none of which by itself is essential.

Animals↗
PubMed2003

Activation of Akt is essential for acetylcholine to trigger generation of oxygen free radicals.

OBJECTIVES: Acetylcholine (ACh) receptor activation in the heart causes mitochondrial production of reactive oxygen species (ROS) that is dependent on mitochondrial K(ATP) channel opening. Recent data show that Akt (also known as protein kinase B) is phosphorylated at its activation site following exposure to ACh. However, since no reliable Akt inhibitor is available, it has not been possible to determine whether Akt activation is an actual step in the protective pathway. METHODS: Cultured rat vascular smooth muscle cells (A7r5) were transiently transfected with a dominant negative Akt (Akt-AAA), thus inhibiting the ability of ACh in these cells to phosphorylate Akt. Transfected cells were identified by co-transfection of enhanced green fluorescent protein (EGFP). ROS production was determined by incubating the cells for 15 min with 1 mM reduced MitoTracker Red which becomes fluorescent only after reacting with ROS. Cells were then triple-washed to remove any voltage-dependent pool of dye and single cell fluorescence was measured. RESULTS: ACh exposure (2 mM) led to a 1.64+/-0.15-fold increase in the average fluorescence over that seen in untreated cells (P=0.002). A similar increase in ROS production occurred after treatment with either the K(ATP) channel opener diazoxide (DIAZ) or the potassium ionophore valinomycin (VAL). Akt-AAA transfection abolished ACh-induced ROS production, but not increased ROS production after treatment with either DIAZ or VAL. CONCLUSION: Thus, at least in the smooth muscle cell model, Akt phosphorylation is an important step in the signal transduction pathway leading from ACh receptor activation to the generation of ROS. The experiments reveal that Akt is positioned between the receptor and the K(ATP) channel in this model.

Acetylcholine↗
PubMed2003

Long-term efficacy, curative potential, and prognostic factors of radiotherapy in primary cutaneous B-cell lymphoma.

PURPOSE: Primary cutaneous B-cell lymphomas (PCBCL) are rare and constitute approximately 5-10% of all cutaneous lymphomas. In the literature, conflicting data exist on the optimal treatment modality regarding the efficacy and the relapse rate after radiotherapy (RT) or polychemotherapy. To evaluate the efficacy of RT, patient data from two centers were analyzed and compared with recent reports in the literature. MATERIALS AND METHODS: Between April 1984 and June 2001, 35 patients with PCBCL, 17 men and 18 women ages 27-86 years, were treated with RT alone (29/35 patients) or postoperative RT (6/35 patients). According to the European Organization for Research and Treatment of Cancer classification for PCBCL, this study group included 21 patients (60%) with primary cutaneous follicle center-cell lymphoma, 7 (20%) with primary cutaneous immunocytoma, 4 (11%) with primary cutaneous large B-cell lymphoma (PCLBCL) of the leg, and 3 (9%) provisional types. RESULTS: A total of 34/35 patients achieved an initial complete response after RT. In one additional patient, RT was interrupted after 16 Gy because of fulminant pneumonia. A total of 11/35 (31%) patients developed cutaneous relapse after a median of 11 months. Three patients developed an in-field response and 8 patients an out-field relapse. After a median follow-up of 52 months, 27/35 patients are alive, whereas 8/35 patients died (three deaths resulting from PCBCL and five unrelated to PCBCL). The 5-year overall survival rate was 75% (95% CI: 55-95%). The 5-year relapse-free survival was 50% (95% CI: 32-68%). Univariate and multivariate analysis revealed disseminated primary lesions in at least two noncontiguous anatomic sites and the histologic subtype PCLBCL as unfavorable prognostic factors. CONCLUSIONS: RT of all visible skin lesions is an effective treatment for localized PCBCL. In patients with cutaneous relapses, RT is an effective treatment option as well.

Adult↗
PubMed2003

P1075 opens mitochondrial K(ATP) channels and generates reactive oxygen species resulting in cardioprotection of rabbit hearts.

We have recently proposed that opening of mitochondrial K(ATP) channels (mitoK(ATP)) acts as a trigger for preconditioning (PC) by causing mitochondria to produce reactive oxygen species (ROS). Controversy exists as to whether the putative sarcolemma-selective K(ATP) channel opener P1075 also opens mitoK(ATP) channels and may be cardioprotective. We purified mitoK(ATP) channels from either rabbit heart, rat heart or rat brain and reconstituted the proteins into liposomes. mitoK(ATP) channels from each of these tissues were opened by P1075 with EC(50) values of 60-90 nM. We next tested whether P1075 causes rabbit cardiomyocytes to produce ROS in a K(ATP)-dependent fashion. Mitochondrial ROS production was monitored by the appearance of fluorescence as reduced MitoTracker Red was oxidized. P1075 (100 microM) led to a 44 +/- 9% increase in ROS generation (P < 0.001 vs. untreated cells), which was similar to the increase seen with 50 microM diazoxide, a selective mitoK(ATP) channel opener (49 +/- 9%, P < 0.001 vs. untreated cells). The effect of P1075 was equally potent at a concentration of 150 nM. The P1075-induced increase in ROS production was blocked by 50 microM glibenclamide (GLI), a non-selective K(ATP) blocker, and by 5-hydroxydecanoate (1 mM), a highly selective mitoK(ATP) blocker (-6 +/- 14% and +4 +/- 12%, respectively; P = n.s). In isolated rabbit hearts, P1075 (150 nM) markedly reduced infarct size compared to control animals (10.6 +/- 8.1% of the area at risk vs. 31.5 +/- 5.6%, P < 0.05). GLI (5 microM) as well as 5-hydroxydecanoate (200 microM) completely blocked P1075's anti-infarct effect (31.7 +/- 9.5% and 27.7 +/- 4.6% infarction, respectively; P = n.s. vs. untreated hearts). These data provide strong evidence that P1075 does open mitoK(ATP) channels and protects the ischemic rabbit heart in a mitoK(ATP)-dependent manner.

Animals↗
PubMed2003

Increased expression of VEGF in glomeruloid reactive angioendotheliomatosis.

Reactive angioendotheliomatosis (RAE) is a very rare disorder characterized by marked proliferation of endothelial cells. It is often associated with infections, such as subacute bacterial endocarditis, but has also been described as an early sign of a developing hematological malignancy. We report the case of a 71-year-old Caucasian female who developed lupus-like RAE lesions. A thorough diagnostic workup and subsequent 3-year clinical follow-up revealed no sign of an underlying infectious or neoplastic disorder. Repetitive serum immunofixations were only once consistent with a monoclonal gammopathy of undetermined significance. In lesional skin, the pronounced bud-like endothelial cell formation was associated with an increased epidermal expression of vascular endothelial growth factor (VEGF), a potent angiogenic mediator. In accordance with the paracrine action of epidermally derived VEGF, vascular endothelial cells in lesional skin revealed increased expression of the VEGF receptor VEGFR-2 (KDR). This case suggests a possible role of epidermally derived VEGF in endothelial cell proliferation in RAE.

Aged↗
PubMed2002

Generation of a novel proteolysis resistant vascular endothelial growth factor165 variant by a site-directed mutation at the plasmin sensitive cleavage site.

Vascular endothelial growth factor (VEGF) is a potent angiogenic mediator in tissue repair. In non-healing human wounds plasmin cleaves and inactivates VEGF165. In the present study, we generated recombinant VEGF165 mutants resistant to plasmin proteolysis. Substitution of Arg110 with Ala110 or Gln110, and Ala111 with Pro111 yielded plasmin-resistant and biologically active VEGF165 mutants. In addition, substitution of Ala111 with Pro111 resulted in a substantial degree of stabilization when incubated in wound fluid obtained from non-healing wounds. These results suggest that the plasmin cleavage site Arg110/Ala111 and the carboxyl-terminal domain play an important role in the mitogenic activity of VEGF165.

Animals↗
PubMed2002

ACh and adenosine activate PI3-kinase in rabbit hearts through transactivation of receptor tyrosine kinases.

Adenosine and acetylcholine (ACh) trigger preconditioning through different signaling pathways. We tested whether either could activate myocardial phosphatidylinositol 3-kinase (PI3-kinase), a putative signaling protein in ischemic preconditioning. We used phosphorylation of Akt, a downstream target of PI3-kinase, as a reporter. Exposure of isolated rabbit hearts to ACh increased Akt phosphorylation 2.62 +/- 0.33 fold (P = 0.001), whereas adenosine caused a significantly smaller increase (1.52 +/- 0.08 fold). ACh-induced activation of Akt was abolished by the tyrosine kinase blocker genistein indicating at least one tyrosine kinase between the muscarinic receptor and Akt. ACh-induced Akt activation was blocked by the Src tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG-1478), an epidermal growth factor receptor (EGFR) inhibitor, suggesting phosphorylation of a receptor tyrosine kinase in an Src tyrosine kinase-dependent manner. ACh caused tyrosine phosphorylation of the EGFR, which could be blocked by PP2, thus supporting this receptor hypothesis. AG-1478 failed to block the cardioprotection of ACh, however, suggesting that other receptor tyrosine kinases might be involved. Therefore, G(i) protein-coupled receptors can activate PI3-kinase/Akt through transactivation of receptor tyrosine kinases in an Src tyrosine kinase-dependent manner.

Acetylcholine↗