Markus Niemeyer

Positron Emission Tomography Using [18F]Galacto-RGD Identifies the Level of Integrin αvβ3 Expression in Man

Purpose: The integrin αvβ3 plays a key role in angiogenesis and tumor cell metastasis and is therefore an important target for new therapeutic and diagnostic strategies. We have developed [18F]Galacto-RGD, a highly αvβ3-selective tracer for positron emission tomography (PET). Here, we show, in man, that the intensity of [18F]Galacto-RGD uptake correlates with αvβ3 expression. Experimental Design: Nineteen patients with solid tumors (musculoskeletal system, n = 10; melanoma, n = 4; head and neck cancer, n = 2; gliobastoma, n = 2; and breast cancer, n = 1) were examined with PET using [18F]Galacto-RGD before surgical removal of the tumor lesions. Snap-frozen specimens (n = 26) were collected from representative areas with low and intense standardized uptake values (SUV) of [18F]Galacto-RGD. Immunohistochemistry was done using the αvβ3-specific antibody LM609. Intensity of staining (graded on a four-point scale) and the microvessel density of αvβ3-positive vessels were determined and correlated with SUV and tumor/blood ratios (T/B). Results: Two tumors showed no tracer uptake (mean SUV, 0.5 ± 0.1). All other tumors showed tracer accumulation with SUVs ranging from 1.2 to 10.0 (mean, 3.8 ± 2.3; T/B, 3.4 ± 2.2; tumor/muscle ratio, 7.7 ± 5.4). The correlation of SUV and T/B with the intensity of immunohistochemical staining (Spearmans r = 0.92; P < 0.0001) as well as with the microvessel density (Spearmans r = 0.84; P < 0.0001) were significant. Immunohistochemistry confirmed lack of αvβ3 expression in normal tissue (benign lymph nodes, muscle) and in the two tumors without tracer uptake. Conclusions: Molecular imaging of αvβ3 expression with [18F]Galacto-RGD in humans correlates with αvβ3 expression as determined by immunohistochemistry. PET with [18F]Galacto-RGD might therefore be used as a new marker of angiogenesis and for individualized planning of therapeutic strategies with αvβ3-targeted drugs.

Patterns of αvβ3 Expression in Primary and Metastatic Human Breast Cancer as Shown by 18F-Galacto-RGD PET

The integrin αvβ3 is a key player in angiogenesis and metastasis. Our aim was to study the uptake patterns of the αvβ3-selective PET tracer 18F-galacto-RGD in invasive ductal breast cancer. Methods: Sixteen patients with primary (n = 12) or metastasized breast cancer (n = 4) were examined with 18F-galacto-RGD PET. Standardized uptake values (SUVs) were derived by region-of-interest analysis, and immunohistochemistry of αvβ3 expression was performed (n = 5). Results: 18F-Galacto-RGD PET identified all invasive carcinomas, with SUVs from 1.4 to 8.7 (mean ± SD, 3.6 ± 1.8; tumor-to-blood and tumor-to-muscle ratios, 2.7 ± 1.6 and 6.2 ± 2.2, respectively). Lymph-node metastases were detected in 3 of 8 patients (mean SUV, 3.3 ± 0.8). SUVs in distant metastases were heterogeneous (2.9 ± 1.4). Immunohistochemistry confirmed αvβ3 expression predominantly on microvessels (5/5) and, to a lesser extent, on tumor cells (3/5). Conclusion: Our results suggest generally elevated and highly variable αvβ3 expression in human breast cancer lesions. Consequently, further imaging studies with 18F-galacto-RGD PET in breast cancer patients for assessment of angiogenesis or planning of αvβ3-targeted therapies are promising.

Subcutaneous Mastectomy With Conservation of the Nipple-Areola Skin: Broadening the Indications

Background:Numerous authors take multiple predictive factors into account to decide whether or not the nipple-areola complex (NAC) can be conserved during mastectomy. These factors include the tumor-nipple distance, tumor size, axillary lymph node status, and lymphovascular invasion. Thus only a very limited percentage of patients can keep their NAC. If the breast gland tissue and all milk ducts can be separated completely from the nipple-areola skin (NA-skin) during subcutaneous mastectomy (SCM), conservation of the NA-skin is feasible even in the case of large, central, and retroareolar tumors. Patients and Methods:From July 2003 to May 2006, we performed 109 SCMs on 96 patients. Total mastectomy was indicated in 94 of these breasts, in 16 because of extensive ductal carcinoma in situ, and 78 breasts with invasive carcinoma required additional axillary dissection resulting in indication for modified radical mastectomy. At least 33 of the breasts had malignancy underneath the skin within the areolar margin (centrally located tumors). After dissection of all the breast tissue, the skin envelope with the areola is turned inside out and all milk ducts and any tissue beneath the areola are precisely dissected under the surgeon’s visual control. Frozen sections and HE histopathologic examination of this retroareolar tissue next to the skin are requested to decide whether the NA-skin can be preserved or not. This study was registered on the www.clinicaltrials.com website and has the following identification number ID: NCT00641628. Results:We found it necessary to dissect the NA-skin in 13 of 109 breasts (12%), altering the procedure to a skin sparing mastectomy. Necrosis of the NA-skin requiring surgical intervention occurred in only 1 of the conserved 96 breasts. After follow-up of 20 to 54 months (median: 34 months), no recurrence within the nipple-areola region was observed. One local recurrence on the chest wall and 1 axillary recurrence were detected. Of 96 patients, 2 developed distant metastases. One death was recorded. Occasionally, partial necrosis of the nipple occurred, with residual depigmentation of the skin but a good or excellent cosmetic result was maintained in most cases. Conclusion:SCM with NAC-skin conservation may be performed according to total mastectomy indications if an intraoperative frozen section (and the corresponding HE histopathology) of the tissue next to the nipple-areola skin is free of tumor. The remaining contraindications for SCM are: extensive tumor involvement of the skin, inflammatory breast cancer, and a clinically suspicious nipple.

Novel markers of mesenchymal stem cells defined by genome-wide gene expression analysis of stromal cells from different sources

Mesenchymal stem cells (MSC) represent a mixture of different cell types, of which only a minority is therapeutically relevant. Surface markers specifically identifying non-differentiated MSC from their differentiated progeny have not been described in sufficient detail. We here compare the gene expression profile of the in vivo bone-forming bone marrow-derived MSC (BM-MSC) with non-bone-forming umbilical vein stromal cells (UVSC) and other non-MSC. Clustering analysis shows that UVSC are a lineage homogeneous cell population, clearly distinct from MSC, other mesenchymal lineages and hematopoietic cells. We find that 89 transcripts of membrane-associated proteins are represented more in cultured BM-MSC than in UVSC. These include previously identified molecules, but also novel markers like NOTCH3, JAG1, and ITGA11. We show that the latter three molecules are also expressed on fibroblast colony-forming units (CFU-F). Both NOTCH3 and ITGA11, but not JAG1, further enrich for CFU-F when combined with CD146, a known marker of cells with MSC activity in vivo. Differentiation studies show that NOTCH3+ and CD146+ NOTCH3+ cells sorted from cultured BM-MSC are capable of adipogenic and osteogenic progeny, while ITGA11-expressing cells mainly show an osteogenic differentiation profile with limited adipogenic differentiation. Our observations may facilitate the study of lineage relationships in MSC as well as facilitate the development of more homogeneous cell populations for mesenchymal cell therapy.

Retrograde autologous priming: is it useful in elective on-pump coronary artery bypass surgery?

BACKGROUND The effect of reduced cardiopulmonary bypass (CPB) prime volume by retrograde autologous priming (RAP) was studied. METHODS Twenty patients undergoing elective coronary artery bypass grafting were randomized to either standard prime (SP) volume (1,602 +/- 202 mL crystalloid prime, n = 10) or RAP (395 +/- 150 mL). RAP was performed by draining crystalloid prime from the arterial and venous lines into a recirculation bag before CPB. Cardiac index, pulmonary vascular resistance index, systemic vascular resistance index, alveolar-arterial oxygen tension difference, pulmonary shunt fraction, extravascular lung water (EVLW), plasma colloid osmotic pressure (COP), crystalloid fluid balance, body weight, and clinical parameters were evaluated perioperatively. RESULTS Demographic data and operative parameters were equal for patients in both groups. During CPB, COP was reduced by 55% in the SP group (9.8 +/- 2.0 vs 21.4 +/- 2.1 mm Hg) and by 41% in the RAP group (12.4 +/- 1.1 vs 20.9 +/- 1.8 mm Hg) (p = 0.008, SP vs RAP group). Compared with preoperatively, EVLW was unchanged in the RAP group 2 hours post-CPB, but it was elevated by 21% in the SP group (p = 0.002, SP vs RAP group). End-CPB crystalloid fluid balance was significantly reduced in the RAP group (1,857 +/- 521 vs 2,831 +/- 637 mL). Postoperative (day 2) weight gain in the SP group (1.5 +/- 1.2 kg, p = 0.021) was absent in the RAP group (0.1 +/- 0.9, NS). Postoperative time to full mobilization was shorter in the RAP group. Postpump cardio-respiratory function did not differ among groups. CONCLUSIONS This small-scale pilot study indicates that by reducing crystalloid fluid administration and fall of COP during CPB, RAP reduces postpump EVLW accumulation and weight gain in uncomplicated coronary artery bypass graft patients with no associated effects on cardio-respiratory function.

Extended indications for nipple-sparing mastectomy.

Abstract:  Ablative breast cancer surgery still includes the routine excision of the nipple‐areola complex (NAC). Nipple‐sparing mastectomy (NSM) removes the breast tissue leaving no or little retroareolar ductal tissue but preserves the entire skin of the breast and the NAC. There is some consensus that NSM might be an oncologically safe option for patients with small and peripherally located tumors and probably for high‐risk patients with prophylactic mastectomy. Several studies demonstrated that NSM may be feasible even in patients with large centrally located tumors or multicentric invasive carcinoma. So far, no generally applicable indications for NSM have been defined because long‐term data are still limited. However, from our review of the literature obtained from a MEDLINE search (2003–2009) we conclude that the range of indications for NSM needs not to be limited to small peripheral tumors or to prophylactic treatment.

Prothrombotic effects of tumor necrosis factor alpha in vivo are amplified by the absence of TNF-alpha receptor subtype 1 and require TNF-alpha receptor subtype 2

IntroductionElevated serum levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) correlate with an increased risk for atherothrombotic events and TNFα is known to induce prothrombotic molecules in endothelial cells. Based on the preexisting evidence for the impact of TNFα in the pathogenesis of autoimmune disorders and their known association with an acquired hypercoagulability, we investigated the effects of TNFα and the role of the TNF receptor subtypes TNFR1 and TNFR2 for arteriolar thrombosis in vivo.MethodsArteriolar thrombosis and platelet-rolling in vivo were investigated in wildtype, TNFR1-/-, TNFR2-/- and TNFR1-/R2-/- C57BL/6 mice using intravital microscopy in the dorsal skinfold chamber microcirculation model. In vitro, expression of prothrombotic molecules was assessed in human endothelial cells by real-time PCR and flow cytometry.ResultsIn wildtype mice, stimulation with TNFα significantly accelerated thrombotic vessel occlusion in vivo upon ferric chloride injury. Arteriolar thrombosis was much more pronounced in TNFR1-/- animals, where TNFα additionally led to increased platelet-endothelium-interaction. TNFα dependent prothrombotic effects were not observed in TNFR2-/- and TNFR1-/R2- mice. In vitro, stimulation of human platelet rich plasma with TNFα did not influence aggregation properties. In human endothelial cells, TNFα induced superoxide production, p-selectin, tissue factor and PAI-1, and suppressed thrombomodulin, resulting in an accelerated endothelial dependent blood clotting in vitro. Additionally, TNFα caused the release of soluble mediators by endothelial cells which induced prothrombotic and suppressed anticoagulant genes comparable to direct TNFα effects.ConclusionsTNFα accelerates thrombus formation in an in vivo model of arteriolar thrombosis. Its prothrombotic effects in vivo require TNFR2 and are partly compensated by TNFR1. In vitro studies indicate endothelial mechanisms to be responsible for prothrombotic TNFα effects. Our results support a more selective therapeutic approach in anticytokine therapy favouring TNFR2 specific antagonists.

Oncostatin M‐Mediated Regulation of KIT‐Ligand‐Induced Extracellular Signal‐Regulated Kinase Signaling Maintains Hematopoietic Repopulating Activity of Lin−CD34+CD133+ Cord Blood Cells

We investigated whether KIT signaling was sufficient to maintain human hematopoietic stem cells in culture or whether, as with murine stem cells, signaling through glycoprotein 130 (gp130) is additionally required. Sorted CD34+CD133+(CD33/CD38/CD71)− cells from human umbilical cord blood (UCB) were cultured in the presence of combinations of KIT‐ligand (KL) and the gp130 stimulating molecule oncostatin M (OSM). We found that OSM increased KL‐induced proliferation, which was accompanied by an expansion in numbers of mature progenitors colony‐forming cells (CFC, CAFCw2). More primitive progenitors, CAFCw6 and long‐term culture‐CFC, were not maintained by KL as a single factor. Although addition of OSM did not improve survival, the KL/OSM combination showed improved maintenance of immature progenitors as well as higher CD34 expression. Similarly, both KL and OSM were required to maintain NOD/SCID‐repopulating activity. In experiments to investigate the underlying mechanism, we found that extracellular signal‐regulated kinase (ERK) and its downstream target p90 ribosomal S6 kinase were activated by KL and downregulated by the inclusion of OSM during stimulation. The p38 mitogen‐activated protein kinase (p38 MAPK) was not modulated by either KL or OSM. Indeed, many of the effects of OSM (increased cell division, maintenance of CFC, and maintenance of high CD34 expression) could be mimicked by using the mitogen‐activated protein kinase kinase inhibitor U0126. More importantly, NOD/SCID‐repopulating activity was preserved in the KL/U0126‐stimulated cells, but not in cells stimulated with a combination of KL and the p38 MAPK inhibitor SB203580. Our results show that the loss of repopulating activity during KL stimulation is counteracted by OSM through the downregulation of ERK pathway signaling.

Hepatitis C Virus Induced Endothelial Inflammatory Response Depends on the Functional Expression of TNFα Receptor Subtype 2

In hepatitis C virus (HCV) infection, morbidity and mortality often result from extrahepatic disease manifestations. We provide evidence for a role of receptors of the innate immune system in virally induced inflammation of the endothelium in vitro and in vivo. Corresponding to the in vitro finding of an HCV-dependent induction of proinflammatory mediators in endothelial cells, mice treated with poly (I:C) exhibit a significant reduction in leukocyte rolling velocity, an increase in leukocyte adhesion to the vessel wall and an increased extravasation of leukocytes. HCV directly promotes activation, adhesion and infiltration of inflammatory cells into the vessel wall by activation of endothelial viral receptors. Poly (I:C) induces the expression of TLR3 in vivo and hereby allows for amplification of all of the aforementioned responses upon viral infection. Proinflammatory effects of viral RNA are specifically mediated by TLR3 and significantly enhanced by tumor necrosis factor alpha (TNFα). HCV-RNA induces the endothelial expression of TNFα and TNFα receptor subtype 2 and we provide evidence that leucocyte adhesion and transmigration in response to activation of viral RNA receptors seem to depend on expression of functional TNFR2. Our results demonstrate that endothelial cells actively participate in immune mediated vascular inflammation caused by viral infections.

The Endothelial Tyrosine Phosphatase SHP-1 Plays an Important Role for Vascular Haemostasis in TNFα-Induced Inflammation In Vivo

Introduction. Inflammation and endothelium-derived superoxides are important pathomechanisms in atherothrombotic diseases. We could previously show that the tyrosine phosphatase SHP-1 acts as a negative regulator in endothelial superoxide production. In this study we investigated the influence of SHP-1 on platelet-endothelium interaction and arterial thrombosis in TNFα-induced endothelial inflammation in vivo. Methods. Arteriolar thrombosis and platelet rolling in vivo were investigated in C57BL/6 mice using intravital microscopy in the dorsal skinfold chamber microcirculation model. Results. Inhibition of SHP-1 by the specific pharmacological inhibitor sodium stibogluconate did not significantly enhance platelet-endothelium interaction in vivo under physiological conditions but led to an augmented fraction of rolling platelets in TNFα-induced systemic inflammation. Accordingly, ferric-chloride-induced arteriolar thrombus formation, which was already increased by SHP-1 inhibition, was further enhanced in the setting of TNFα-induced inflammation. Platelet aggregation in vitro as well as ex vivo was not influenced by SHP-1-inhibition. In cultured endothelial cells, sodium stibogluconate increased TNFα-induced surface expression of p-selectin and von Willebrand factor. Additionally, TNFα increased SHP-1 activity and protein expression. Conclusions. The endothelial tyrosine phosphatase SHP-1 plays an important role for vascular hemostasis in vivo, which is crucial in TNFα-induced endothelial inflammation where it may serve as an autoinhibitory molecule to prevent excess inflammatory response and thrombus formation.