Endothelial Cells Of Small Vessels Research Articles

Regulation of Tumor Vascular Microenvironment by Nestin and Fms-related Tyrosine Kinase 1 (FLT1) and Their Prognostic Significance in Renal Cell Carcinoma.

Hypervascularity is a characteristic feature of renal cell carcinoma (RCC) and microvessel density (MVD) predicts tumor metastasis. Nestin is a stem cell marker that is expressed in proliferating endothelial cells and newly formed vessels and Fms-related tyrosine kinase 1 (FLT1) is a proangiogenic factor. This study aimed to evaluate the expression of Nestin and FLT1 in RCC and their prognostic impact. This retrospective study included sixty cases of RCC after obtaining ethical approval. Sections were immunohistochemically stained by Nestin and FLT1 then their expressions were compared to different clinicopathological parameters. MVD was evaluated using Nestin and CD34 and compared to the different parameters. Nestin was expressed mainly in endothelial cells of small vessels in 65% of cases while FLT1 was expressed in tumor and endothelial cells in 73.3% of cases. Their expressions were significantly associated with aggressive tumor parameters including larger tumors, high-grade tumors, wider tumor extension, and advanced stage. Moreover, Nestin expression was significantly associated with metastasis. MVD evaluated by Nestin showed more associations with larger tumors, high-grade tumors, wider tumor extension, advanced stage, and metastasis than MVD measured by CD34. Nestin and FLT1 positivity and high MVD measured by Nestin were significantly associated with short overall survival. Nestin and FLT1 expressions in RCC may be associated with aggressive tumor features and short patients' overall survival. MVD evaluated by Nestin may be correlated with tumor progression and metastasis. Nestin and FLT1 may be used as prognostic biomarkers in RCC.

Abstract 5206: High level STING expression in tumor and inflammatory cells is linked to microsatellite instability and favorable tumor parameters in a cohort of 1,900 colorectal cancer patients

Abstract Background: The stimulator of interferon genes (STING) is a transmembrane protein expressed on a variety of cell types including epithelial, inflammatory (lymphocytes and macrophages), and endothelial cells. STING expression has been described at variable levels in various tumor types where it might enhance anti-tumoral effects of the immune system and impact tumor angiogenesis. STING activating targeted therapies are currently evaluated in preclinical studies. However, the clinical significance of STING expression in tumor cells as compared to tumor-associated inflammatory cells is not fully resolved. Methods: To evaluate the potential clinical significance of STING expression in different cell types of colorectal cancer (CRC) tissue (tumor cells, lymphocytes, macrophages and endothelial cells), more than 1,900 patients were analyzed by multiplex fluorescence immunohistochemistry (CKpan, STING, CD45, CD68 and CD31) in a tissue microarray format (TMA) in combination with a deep-learning based algorithm for automated cell detection. Results: STING expression on colorectal cancer cells varied markedly. Of 1,905 evaluable cancers, 601 (31.5%) showed a strong expression in virtually all tumor cells and 428 (22.5%) lacked STING staining while 478 (25.1%) showed a weak, and 398 (20.9%) a moderate staining. High STING expression on tumor cells was associated with microsatellite instability (MSI, p<0.0001), low tumor stage (p=0.0013), absence of nodal metastasis (pN0, p=0.0003) and tumor localization in the right colon (p<0.0001). A multivariate analysis revealed that these associations were solely driven by the tight relationship between STING expression in tumor cells and MSI. Accordingly, subgroups of tumors with and without MSI lacked significant associations between STING expression on tumor cells and pT or pN categories. While the total number of lymphocytes and macrophages were related to MSI, low pT, and pN0, there were significant associations between a high percentage of STING positive macrophages and lymphocytes to low pT stage (p<0.0001 each) and absence of nodal metastases for high percentage of STING positive macrophages (p=0.0033). STING was regularly expressed on endothelial cells of small vessels. Although the intensity appeared to be variable, there was no link to tumor phenotype. Conclusion: In summary, the significant link between high STING expression on tumor cells, lymphocytes and macrophages with a favorable tumor phenotype supports the concept of STING representing an enhancer of anti-tumoral effects of the immune system. Citation Format: Elena Bady, Zhihao Huang, Maximilian Lennartz, Claudia Hube-Magg, Julia Ebner, Frank Jacobsen, Stefan Steurer, Christian Bernreuther, Till S. Cauditz, Andreas H. Marx, Till Krech, Eike Burandt, Guido Sauter, Ronald Simon, Sarah Minner, Jan Hendrik Mueller. High level STING expression in tumor and inflammatory cells is linked to microsatellite instability and favorable tumor parameters in a cohort of 1,900 colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5206.

Clinical manifestations and imaging and pathological features of giant cell angioblastoma: Report of four cases and literature review.

Giant cell angioblastoma is a relatively rare vasogenic tumour. To date, studies on its clinical manifestations, imaging characteristics, pathological features, and prognosis are extremely limited and unknown, with only a few cases recorded. In this study, four cases of giant cell angioblastoma confirmed by pathological examination were reported to improve our understanding and deep exploration of the tumour spectrum. All cases in our study were male, including two adults and two boys. The lesions were located in the lower segment of the femur, medial condyle of the femur, knee joint, and popliteal fossa. Regarding the imaging characteristics, two patients with lesions in bone showed bone destruction, while the other two had lesions that invaded soft tissues, showing irregular, abnormal signal shadows and obvious enhancement. Histopathological analysis revealed that the nodular tumour tissue was mainly composed of oval and spindle cells, with varying numbers of osteoclast-like multinucleated giant cells, and the interstitial tissues were often filled with blood vessels of different sizes. The immunophenotype demonstrates that endothelial cells of small vessels in nodules expressed CD31, SMA, and ERG, while osteoclast-like multinucleated giant cells and histiocytes expressed CD68 and CD163, and the surrounding cells expressed SMA. All four patients were treated with surgical resection. One of them relapsed 1 month after surgery and received a second surgical resection. No distant metastasis or death occurred during the follow-up period. This study indicates that giant cell angioblastoma is a local invasive vascular tumour that can develop both in children and adults with skin, mucous membrane, soft tissue, and bone involvement. Imaging characteristics show bone destruction and irregular, abnormal signal shadows; in addition, obvious pathological morphological features can be observed. Currently, the treatment is mainly surgical resection, and interferons may be used as adjuvant chemotherapy.

Polarization of infiltrating macrophages in the outer annulus fibrosus layer associated with the process of intervertebral disc degeneration and neural ingrowth in the human cervical spine

BACKGROUND CONTEXTAs no infiltrating macrophages exist in healthy discs, understanding the role of infiltrating macrophages including their polarity (M1 and M2 phenotypes) in intervertebral discs (IVDs) is important in the assessment of the pathomechanisms of disc degeneration. PURPOSETo determine the relationship between infiltrating macrophage polarization and the progression of human cervical IVD degeneration. STUDY DESIGNHistopathological study using harvested human cervical IVDs. METHODSIVDs collected during anterior cervical decompression from 60 patients were subjected to immunostaining and immunoblotting. The samples were classified as type 0–3 according to the percentage of CD16- and CD206-positive cells to CD68-positive cells in the outer annulus fibrosus layer. The number of vessels and nerve fibers and the severity of chronic inflammation with a focus on inflammatory cell infiltration, fibrosis, and capillary proliferation were also assessed. RESULTSThe number of CD16-positive cells was the highest in type 2 IVDs, and was suppressed following the infiltration of CD206-positive cells. The degree of chronic inflammation was significantly higher in type 2 and type 3 IVDs, and the number of nerve fibers was significantly higher in type 3 IVDs. The endothelial cells of small vessels were positive for nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 expression. Staining for tropomyosin receptor kinase (Trk)-A, Trk-B, and Trk-C was positive in aberrant fibers. In immunoblot analysis, the expression levels of these neurotrophic factors and receptors were significantly higher in type 2 and 3 IVDs. CONCLUSIONSThe polarity of macrophages around newly developed microvasculature might be altered with cervical IVD degeneration. A higher number of infiltrating M1 macrophages around the vessels was associated with chronic inflammation; however, their number got suppressed following the infiltration of M2 macrophages. The expression of neurotrophins in the capillaries of small vessels might contribute to neural ingrowth into degenerated IVDs. CLINICAL SIGNIFICANCEClarifying macrophages polarity change around new microvasculature associated with progression of IVD degeneration could enhance our understanding of the underlying mechanisms of neural ingrowth into degenerated IVDs and lead to development of a novel therapeutic target for prevention of IVD.

Re-evaluation of the immunohistochemical distribution of isoforms of nitric oxide synthase in the human prostate: A light and electron microscopical study.

Up until today, there are still uncertainties regarding the occurrence of isoforms of the nitric oxide synthase (eNOS, nNOS) in the human prostate. While nNOS was exclusively seen in slender nerve fibres branching within the transition zone, eNOS was reported in glandular structures and also in small vessels interspersing the tissue. This study aimed to re-evaluate by means of light and electron microscopy (LM, EM), the distribution of eNOS and nNOS in the transition zone of the human prostate. Tissue specimens were obtained from 16 patients who underwent surgery for pelvic malignancies. Using specific antibodies in conjunction with advanced fixation and staining procedures, the occurrence of eNOS and nNOS was investigated. nNOS was detected in nerve fibres interspersing the tissue and was also seen in glandular structures. EM revealed that in glandular epithelial cells immunoreaction for nNOS was limited to the cytoplasmic compartment. Vascular endothelial cells of small vessels transversing glandular structures significantly stained for eNOS, while epithelial layers of prostatic glandules appeared free of eNOS. The results implicate that, in the prostate, nNOS is a mediator of stromal and glandular tissue function, and counteract the assumption of eNOS activity in glandular epithelial cells as a source of NO synthesis.

Complement in Sickle Cell Disease: Are We Ready for Prime Time?

Sickle cell disease (SCD) is a widely spread inherited hemoglobinopathy that includes a group of congenital hemolytic anemias, all characterized by the predominance of sickle hemoglobin (HbS). Its features are anemia, predisposal to bacterial infections and complications such as vaso-occlusive crisis (VOC) or delayed hemolytic transfusion reaction (DHTR), which lead to increased rate of morbidity and mortality even in the era of hydroxyurea. The interaction between sickle cells, neutrophils, platelets or endothelial cells in small vessels results in hemolysis and has been considered the disease’s main pathophysiological mechanism. Complement activation has been reported in small cohorts of SCD patients, but the governing mechanism has not been fully elucidated. This will be important to predict the patient group that would benefit from complement inhibition. Until now, eculizumab-mediated complement inhibition has shown beneficial effects in DHTR, with limited reports in patients with VOC. In the meantime, several innovative agents are under clinical development Our state-of-the-art review summarizes current data on 1) complement activation in SCD both in steady state and crisis, 2) underlying mechanisms of complement over-activation for the clinician in the context of SCD, 3) actions of hydroxyurea and new therapeutic approaches including indirect involvement in complement activation, and 4) novel paradigms in complement inhibition.

Incontinentia Pigmenti Associated with Aplasia Cutis Congenita in a Newborn Male with Klinefelter Syndrome: Is the Severity of Neurological Involvement Linked to Skin Manifestations?

We report a rare case of a newborn male affected by incontinentia pigmenti, Klinefelter syndrome, and aplasia cutis congenita, who developed severe cutaneous, neurological, and ophthalmological manifestations. Genetic analysis showed the presence of the common mutation of NEMO (exon 4–10 deletion), Klinefelter syndrome karyotype (47 XXY), and random X inactivation. This is in accordance with the severity of involvement of the affected tissues (skin, central nervous system, and retina). Indeed, the patient developed typical skin lesions all over the body, except the head. Equally, multiple lesions diffusely involving both the cortical grey matter and subcortical white matter of the cerebellum and cerebral hemispheres were observed. Discussing current knowledge about the etiopathogenesis of skin and brain lesions in incontinentia pigmenti, our case seems to support the proapoptotic origin of central nervous system involvement. Possibly, incontinentia pigmenti patients suffer an impaired protection against apoptosis at the level of cerebral endothelial cells of small vessels, leading to vascular damage and subsequent ischemic brain lesions.

The Vasodilating Effect and Angiotensin Converting Enzyme Inhibition Activity of Three Dietary Flavonols: Comparsion between Myricetin, Quercetin and Morin, <i>in vitro</i>

The aim of this study is to compare the vasodiating effects and angiotensin converting enzyme inhibition activity of three dietary flavonols, myricetin, quercetin and morin, in vitro. The three flavonols ranging from 0 to 200 μM show no cytotoxicity to small vessel endothelial cells (SVEC). For vasodilative assay, of the three flavonols, myricetin showed the greatest induction in the levels of nitric oxide (NO) and prostacyclin (PGI2), and the expression of cyclooxygenase (COX-2). Myricetin, quercetin and morin enhanced the expression of eNOS, but no significant differences (p > 0.05) were found between three flavonols. Myricetin, quercetin and morin showed marked inhibitory effect on reactive oxygen species (ROS) production in H2O2-induced SVEC cells. In addition, myricetin and quercetin showed relatively higher inhibitory activity on ACE activity than morin. Taken together, of the three flavonols, myricetin with the substitution by more hydroxyl groups in the flavonol structure enhanced vasodilating activity. Enzyme kinetic analysis showed myricetin is a non-competitive inhibitor of ACE. These structure-function relationships facilitate the design of new antihypertensive drugs based on flavonols.

Pericytes as a Source of Osteogenic Cells in Bone Fracture Healing.

Pericytes are mesenchymal cells that surround the endothelial cells of small vessels in various organs. These cells express several markers, such as NG2, CD146, and PDGFRβ, and play an important role in the stabilization and maturation of blood vessels. It was also recently revealed that like mesenchymal stem cells (MSCs), pericytes possess multilineage differentiation capacity, especially myogenic, adipogenic, and fibrogenic differentiation capacities. Although some previous studies have reported that pericytes also have osteogenic potential, the osteogenesis of pericytes can still be further elucidated. In the present study, we established novel methods for isolating and culturing primary murine pericytes. An immortalized pericyte line was also established. Multilineage induction of the pericyte line induced osteogenesis, adipogenesis, and chondrogenesis of the cells in vitro. In addition, pericytes that were injected into the fracture site of a bone fracture mouse model contributed to callus formation. Furthermore, in vivo pericyte-lineage-tracing studies demonstrated that endogenous pericytes also differentiate into osteoblasts and osteocytes and contribute to bone fracture healing as a cellular source of osteogenic cells. Pericytes can be a promising therapeutic candidate for treating bone fractures with a delayed union or nonunion as well as bone diseases causing bone defects.

Assessment of Angiogenic Markers and Female Sex Hormone Receptors in Pregnancy Tumor of the Gingiva

Oral pregnancy tumors (OPTs) arise on the inflamed gingiva of women after the first trimester of pregnancy. The expression of angiogenic markers and female hormone receptors was assessed. Immunohistochemistry was used to analyze the expression of estrogen and progesterone receptors and the expression of angiogenic factors, such as vascular endothelial growth factor (VEGF) and its receptor, fibroblast growth factor (FGF), and hypoxia inducible factors 1α and 3α (HIF1α and HIF3α). Experimental groups included 9 OPTs, 10 oral pyogenic granulomas from nonpregnant women of the same age, and 9 oral pyogenic granulomas from postmenopausal women. VEGF expression in stromal histiocytes and endothelial cells of small vessels was positively correlated in the OPT group (P < .05 by χ(2) test). VEGF receptor also was overexpressed in stromal histiocytes and endothelial cells of OPTs compared with oral pyogenic granulomas from nonpregnant and postmenopausal women (P < .005 by χ(2) test). No correlation was detected among estrogen and progesterone receptors, FGF and HIF1α and HIF3α (ER and PgR respectively) in the 3 experimental groups. VEGF-associated angiogenesis is most likely involved in the pathogenesis of the lesion. These results imply that local inhibition of VEGF activity could be an adjuvant therapeutic approach for OPTs to control hemorrhage, which can be massive at the surgical excision of such lesions during pregnancy.

Photodynamic therapy involves an antiangiogenic mechanism and is enhanced by ferrochelatase inhibitor in urothelial carcinoma

The purpose of the present study was to investigate the mechanism of photodynamic therapy (PDT) supplemented with exogenously added 5-aminolevulinic acid (ALA) on human urothelial cancer (UC). Moreover, we aimed to determine whether the therapeutic effects of ALA-based PDT (ALA-PDT) for UC could be enhanced by deferoxamine (DFX), an inhibitor of ferrochelatase. The efficiency of ALA-PDT on these cells was analyzed using flow cytometry and the type of cell death was also assessed. The ALA-PDT promoting effect of DFX was examined on both UC cells and human umbilical vein endothelial cells (HUVEC). The ALA-PDT decreased levels of mitochondrial membrane potential and induced cell death mainly via apoptosis in these cells. Moreover, inhibition of ferrochelatase by DFX led to an increase of protoporphyrin IX (PpIX) accumulation and enhanced the effect of ALA-PDT on UC cells. We further investigated the effect of DFX on in vivo PDT with a tumor-bearing animal model and found that DFX efficiently enhanced tumor cell apoptosis. ALA-PDT induced death of neovascular endothelial cells in tumors but did not affect small vessel endothelial cells in normal tissues surrounding the tumor. Furthermore, DFX enhanced inhibition of neovascularization. These results demonstrated ALA-PDT dominantly induced apoptosis over necrosis by direct action on UC as well as via antiangiogenic action on neovacular endothelial cells, suggesting that the therapeutic damage by ALA-PDT could be kept to a minimum in the surrounding normal tissues. In addition, increased accumulation of PpIX by DFX could enhance this effectiveness of ALA-PDT.

Thy-1 (CD90) Is an Interacting Partner for CD97 on Activated Endothelial Cells

Leukocyte recruitment in response to inflammatory signals is governed, in part, by binding to Thy-1 (CD90) on activated endothelial cells (EC). In this study, we characterized the adhesion G-protein coupled receptor CD97, present on peripheral myeloid cells, as a novel interacting partner for Thy-1. CD97 was upregulated on polymorphonuclear cells (PMNC) of patients with psoriasis. In psoriatic skin lesions, CD97(+) myeloid cells colocalized with Thy-1(+) EC of small vessels in microabscesses, suggesting an interaction between CD97 and Thy-1 that was further examined by adhesion and protein-binding assays. PMNC and cell lines stably overexpressing CD97 adhered specifically to Thy-1(+)-activated human dermal EC, Thy-1(+) CHO cells, and immobilized Thy-1 protein. Binding of the CD97(+) CHO clones correlated with their CD97 expression level. Soluble CD97 bound specifically to immobilized Thy-1 protein, as well as Thy-1(+)-activated EC and CHO cells. In all assays, cellular adhesion or protein binding was blocked partially by CD97 and Thy-1-blocking mAb. Our data suggested that CD97 interacts via its stalk with Thy-1 because mAb directed to the stalk of CD97 showed stronger blocking compared with mAb to its epidermal growth factor-like domains, and binding was calcium independent. Moreover, soluble CD97 without the stalk and soluble EMR2, containing highly homologous epidermal growth factor-like domains but a different stalk, failed to bind. In summary, binding of leukocytes to activated endothelium mediated by the interaction of CD97 with Thy-1 is involved in firm adhesion of PMNC during inflammation and may play a role in the regulation of leukocyte trafficking to inflammatory sites.

Subcellular localization and distribution of the reduced folate carrier in normal rat tissues

The reduced folate carrier (Rfc1; Slc19a1) mediated transport of reduced folates and antifolate drugs such as methotrexate (MTX) play an essential role in physiological folate homeostasis and MTX cancer chemotherapy. As no systematic reports are as yet available correlating Rfc1 gene expression and protein levels in all tissues crucial for folate and antifolate uptake, storage or elimination, we investigated gene and protein expression of rat Rfc1 (rRfc1) in selected tissues. This included the generation of a specific anti-rRfc1 antibody. Rabbits were immunised with isolated rRfc1 peptides producing specific anti-rRfc1 antiserum targeted to the intracellular C-terminus of the carrier. Using RT-PCR analysis, high rRfc1 transcript levels were detected in colon, kidney, brain, thymus, and spleen. Moderate rRfc1 gene expression was observed in small intestine, liver, bone marrow, lung, and testes whereas transcript levels were negligible in heart, skeletal muscle or leukocytes. Immunohistochemical analyses revealed strong carrier expression in the apical membrane of tunica mucosa epithelial cells of small intestine and colon, in the brush-border membrane of choroid plexus epithelial cells or in endothelial cells of small vessels in brain and heart. Additionally, high rRfc1 protein levels were localized in the basolateral membrane of renal tubular epithelial cells, in the plasma membrane of periportal hepatocytes, and sertoli cells of the testes. Taken together, our results demonstrated that rRfc1 is expressed almost ubiquitously but to very different levels. The predominant tissue distribution supports the essential role of Rfc1 in physiological folate homeostasis. Moreover, our results may contribute to understand antifolate pharmacokinetics and selected organ toxicity associated with MTX chemotherapy.

Intravascular lymphomatosis and intracerebral haemorrhage

Intravascular lymphomatosis (IVL) is a rare, malignant B- or T-cell lymphoma with remarkable affinity for the endothelial cells of small vessels, particularly within the skin and central nervous system. It is a disease that mimics several neurological disorders, particularly those of cerebrovascular ischemic origin. The prognosis is generally poor, with a rapidly fatal outcome. As a result the diagnosis is often made at post-mortem. We report a rare case of a 73-year-old patient with IVL complicated by intracerebral haemorrhage. In literature two cases of systemic IVL complicated by intracerebral haemorrhage have been reported, but they presented initially with a disseminated intravascular coagulation (DIC). This is the first case of brain IVL complicated by intracerebral haemorrhage not associated to DIC. Increasing awareness of this disease as a differential diagnosis to a common clinical presentation may lead to more opportunities to evaluate new diagnostic and treatment approaches.

Stromal vascular progenitors in adult human adipose tissue.

The in vivo progenitor of culture-expanded mesenchymal-like adipose-derived stem cells (ADSC) remains elusive, owing in part to the complex organization of stromal cells surrounding the small vessels, and the rapidity with which adipose stromal vascular cells adopt a mesenchymal phenotype in vitro. Immunohistostaining of intact adipose tissue was used to identify three markers (CD31, CD34, and CD146), which together unambiguously discriminate histologically distinct inner and outer rings of vessel-associated stromal cells, as well as capillary and small vessel endothelial cells. These markers were used in multiparameter flow cytometry in conjunction with stem/progenitor markers (CD90 and CD117) to further characterize stromal vascular fraction (SVF) subpopulations. Two mesenchymal and two endothelial populations were isolated by high speed flow cytometric sorting, expanded in short term culture, and tested for adipogenesis. The inner layer of stromal cells in contact with small vessel endothelium (pericytes) was CD146+/alpha-SMA+/CD90+/-/CD34-/CD31-; the outer adventitial stromal ring (designated supra adventitial-adipose stromal cells, SA-ASC) was CD146-/alpha-SMA-/CD90+/CD34+/CD31-. Capillary endothelial cells were CD31+/CD34+/CD90+ (endothelial progenitor), whereas small vessel endothelium was CD31+/CD34-/CD90- (endothelial mature). Flow cytometry confirmed these expression patterns and revealed a CD146+/CD90+/CD34+/CD31- pericyte subset that may be transitional between pericytes and SA-ASC. Pericytes had the most potent adipogenic potential, followed by the more numerous SA-ASC. Endothelial populations had significantly reduced adipogenic potential compared with unsorted expanded SVF cells. In adipose tissue, perivascular stromal cells are organized in two discrete layers, the innermost consisting of CD146+/CD34- pericytes, and the outermost of CD146-/CD34+ SA-ASC, both of which have adipogenic potential in culture. A CD146+/CD34+ subset detected by flow cytometry at low frequency suggests a population transitional between pericytes and SA-ASC.

Novel Pleiotropic Effects of Thrombin in Regulation of the Metastatic Potential of Human Rhabdomyosarcoma (RMS) Cells.

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of adolescents and children that frequently infiltrates bone marrow (BM). We identified chemokines and growth factors [i.e., stromal derived factor (SDF)-1, hepatocyte growth factor (HGF)] that play important roles in RMS metastasis (Blood 2002; 100:2597, Cancer Res. 2003; 63:7926,Cancer Res. 2007; 67:2131). However, novel evidence indicates that the metastatic process for many tumors may be modulated by components of the coagulation cascade (e.g., thrombin, activated platelets). Thus, we became interested in the role of coagulation cascade in modulating metastasis of RMS cells. We learned that RMS cells express tissue factor (TF) and thus may activate coagulation by generating thrombin. Thrombin activated in the tumor microenvironment activates platelets that release microvesicles. These interact with RMS cells and/or directly stimulate PAR 1–4 receptors that are expressed on RMS cells. Focusing on these effects, 9 human RMS cells lines were employed for our studies. We learned that platelet-derived microvesicles (PMVs) transfer several platelet integrin receptors to RMS cells (e.g., a2b3) that are important for interaction with the endothelium, thus increasing their adhesive potential to endothelial cells, fibronectin, and fibrinogen. More importantly, RMS cells covered with PMVs showed higher metastatic potential after intravenous injection into severe combined immunodeficient (SCID) mice. This could be explained by enhanced adhesiveness of PMV-covered RMS cells to endothelial cells in small vessels. On the other hand, PMVs also directly chemoattracted RMS cells and activated MAPKp42/44 and AKT. Since we found that RMS cell lines express functional PAR 1–4 receptors, we become interested in the thromin-mediated responses of these cells. In RMS, we observed cells stimulated by thrombin phosphorylation of MAPKp42/44 and p38, but not AKT. However, we did not observe any effect from thrombin on proliferation and survival of RMS cells. To our surprise, thrombin decreased RMS chemotactic responses to SDF-1, HGF, and PMVs. Thrombin also decreased adhesion of RMS cells to fibronectin and laminin. In conclusion, we demonstrate for first time that RMS-expressed TF activates prothrombin and that thrombin is a novel, underappreciated, pro-metastatic factor for these cells. Activated in tumor proximity by thrombin, platelets release PMVs that chemoattract and transfer several platelet-derived receptors/adhesion molecules to RMS cells that are crucial for adhesion/interaction with the endothelium. Conversely, by decreasing the responsiveness of RMS cells to local chemoattractants and decreasing adhesiveness of RMS cells, thrombin promotes their release from the primary tumor into circulation. Consequently, RMS cells that are covered by PMVs release into circulation and respond to chemoattractants in distant organs for metastasis.

Expression of multi-drug resistance 1 mRNA in human and rodent tissues: reduced levels in Parkinson patients

The membrane transporter multi-drug resistance 1 (MDR1, P-gp) regulates the bioavailability of endogenous and exogenous compounds and has been implicated in disorders such as Parkinson's disease, cancer, epilepsy, human immunodeficiency virus disease, and inflammatory bowel disease. To promote further understanding of the role of MDR1 in disease, we have characterized cellular MDR1 mRNA expression in post-mortem human and fresh-frozen Sprague-Dawley rat tissues by using radioactive oligonucleotide probe in situ hybridization. We report MDR1 mRNA in human and rat endothelial cells of small vessels in the brain and pia mater. Mdr1 mRNA is also expressed in the blood vessel walls of rat sensory dorsal root and sympathetic ganglia. In peripheral tissues, we have observed MDR1 mRNA in human and rat liver and renal tubules and in human adrenal cortex and the epithelial lining of rat intestine. In female and male reproductive tissues of rat, strong gene activity has been found in steroid-hormone-synthesizing cells. Quantification of MDR1 mRNA in human striatum has revealed reduced levels in Parkinson patients compared with control individuals. The high expression of MDR1 mRNA in blood vessels of the nervous system, in tissues involved in absorption and excretion, and in tissues forming barriers to the environment support the physiological role of MDR1 as a regulator of intracellular levels of endogenous and exogenous compounds.

Gastrointestinal Dysmotility in Mitochondrial Neurogastrointestinal Encephalomyopathy Is Caused by Mitochondrial DNA Depletion

Chronic intestinal pseudo-obstruction is a life-threatening condition of unknown pathogenic mechanisms. Chronic intestinal pseudo-obstruction can be a feature of mitochondrial disorders, such as mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a rare autosomal-recessive syndrome, resulting from mutations in the thymidine phosphorylase gene. MNGIE patients show elevated circulating levels of thymidine and deoxyuridine, and accumulate somatic mitochondrial DNA (mtDNA) defects. The present study aimed to clarify the molecular basis of chronic intestinal pseudo-obstruction in MNGIE. Using laser capture microdissection, we correlated the histopathological features with mtDNA defects in different tissues from the gastrointestinal wall of five MNGIE and ten control patients. We found mtDNA depletion, mitochondrial proliferation, and smooth cell atrophy in the external layer of the muscularis propria, in the stomach and in the small intestine of MNGIE patients. In controls, the lowest amounts of mtDNA were present at the same sites, as compared with other layers of the gastrointestinal wall. We also observed mitochondrial proliferation and mtDNA depletion in small vessel endothelial and smooth muscle cells. Thus, visceral mitochondrial myopathy likely causes gastrointestinal dysmotility in MNGIE patients. The low baseline abundance of mtDNA molecules may predispose smooth muscle cells of the muscularis propria external layer to the toxic effects of thymidine and deoxyuridine, and exposure to high circulating levels of nucleosides may account for the mtDNA depletion observed in the small vessel wall.

Cytomegalovirus Prostatitis: A Series of 4 Cases

Cytomegalovirus (CMV) prostatitis is very rare with only 1 report of biopsy-proven CMV prostatitis in the literature. The authors report 4 cases, 3 detected on needle biopsy and 1 detected on transurethral resection. Patients were 36, 41, 48, and 71 years old. All patients were immunosuppressed, including 1 with AIDS and 3 undergoing immunosuppressive therapy following organ transplantation. CMV inclusions were seen in secretory cells of the prostatic glands, endothelial cells of small vessels, and prostatic stromal cells associated with a dense lymphoid inflammation. Only very rarely is CMV prostatitis detected on clinical specimens, typically in immunosuppressed hosts undergoing immunosuppressive therapy following organ transplantation. Patients with CMV prostatitis may harbor multiple infections or have other serious medical conditions adversely affecting their prognosis.

Anti-angiogenic activity of sesterterpenes; natural product inhibitors of FGF-2-induced angiogenesis

Angiogenesis, the growth of new blood vessels from the pre-existing vasculature is of physiological and pathological importance. We have investigated the anti-angiogenic potential of two naturally occurring sesterterpenes, leucosesterterpenone (compound 1) and leucosterlactone (compound 2) isolated from the Himalayan plant Leucosceptrum canum and identified as having biological activity in preliminary screening. Compound 1 inhibited fibroblast growth factor-2-induced proliferation, migration in a wounding assay, chemotaxis and tube formation with small vessel (human dermal) and large vessel (bovine aortic) endothelial cells while compound 2 was largely inactive. Both compounds were also active in an in vivo angiogenic model using the chick chorioallantoic membrane. Neither compounds showed inhibitory activity in the absence of fibroblast growth factor-2. We were able to demonstrate in a binding assay that compounds 1 and 2 bound to the fibroblast growth factor-2 receptor-1 with IC(50) values of 1.4 +/- 0.956 and 132.47 +/- 7.90 muM, respectively, with a concomitant down regulation of phosphorylated ERK1/2 but did not bind to receptor-2. Compound 1 was less hydrophobic than compound 2 and this may contribute to its increased activity. Compound 1 is a new addition to the small number of inhibitors of fibroblast growth factor-2-induced angiogenesis. The compound was a specific inhibitor in that it had no effect on vascular endothelial growth factor or epithelial growth factor-induced angiogenesis. Since angiogenesis is essential for tumour development we conclude that these compounds may have potential as anti-tumour agents.