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Supplementary Figure S3. Association of the cell cycle/immune signature with B, lactate dehydrogenase levels.

Purpose: Histone deacetylase (HDAC) inhibitors have shown promising clinical activity in the treatment of hematologic malignancies, but their activity in solid tumor indications has been limited. Most HDAC inhibitors in clinical development only transiently induce histone acetylation in tumor tissue. Here, we sought to identify a second-generation class I HDAC inhibitor with prolonged pharmacodynamic response in vivo, to assess whether this results in superior antitumoral efficacy.Experimental Design: To identify novel HDAC inhibitors with superior pharmacodynamic properties, we developed a preclinical in vivo tumor model, in which tumor cells have been engineered to express fluorescent protein dependent on HDAC1 inhibition, thereby allowing noninvasive real-time evaluation of the tumor response to HDAC inhibitors.Results:In vivo pharmacodynamic analysis of 140 potent pyrimidyl-hydroxamic acid analogues resulted in the identification of JNJ-26481585. Once daily oral administration of JNJ-26481585 induced continuous histone H3 acetylation. The prolonged pharmacodynamic response translated into complete tumor growth inhibition in Ras mutant HCT116 colon carcinoma xenografts, whereas 5-fluorouracil was less active. JNJ-26481585 also fully inhibited the growth of C170HM2 colorectal liver metastases, whereas again 5-fluorouracil/Leucovorin showed modest activity. Further characterization revealed that JNJ-26481585 is a pan-HDAC inhibitor with marked potency toward HDAC1 (IC50, 0.16 nmol/L).Conclusions: The potent antitumor activity as a single agent in preclinical models combined with its favorable pharmacodynamic profile makes JNJ-26481585 a promising second-generation HDAC inhibitor. The compound is currently in clinical studies, to evaluate its potential applicability in a broad spectrum of both solid and hematologic malignancies. (Clin Cancer Res 2009;15(22):684151)

Purpose: The association of tumor gene expression profiles with progression-free survival (PFS) outcomes in patients with BRAFV600-mutated melanoma treated with vemurafenib or cobimetinib combined with vemurafenib was evaluated.Experimental Design: Gene expression of archival tumor samples from patients in four trials (BRIM-2, BRIM-3, BRIM-7, and coBRIM) was evaluated. Genes significantly associated with PFS (P < 0.05) were identified by univariate Cox proportional hazards modeling, then subjected to unsupervised hierarchical clustering, principal component analysis, and recursive partitioning to develop optimized gene signatures.Results: Forty-six genes were identified as significantly associated with PFS in both BRIM-2 (n = 63) and the vemurafenib arm of BRIM-3 (n = 160). Two distinct signatures were identified: cell cycle and immune. Among vemurafenib-treated patients, the cell-cycle signature was associated with shortened PFS compared with the immune signature in the BRIM-2/BRIM-3 training set [hazard ratio (HR) 1.8; 95% confidence interval (CI), 1.3–2.6, P = 0.0001] and in the coBRIM validation set (n = 101; HR, 1.6; 95% CI, 1.0–2.5; P = 0.08). The adverse impact of the cell-cycle signature on PFS was not observed in patients treated with cobimetinib combined with vemurafenib (n = 99; HR, 1.1; 95% CI, 0.7–1.8; P = 0.66).Conclusions: In vemurafenib-treated patients, the cell-cycle gene signature was associated with shorter PFS. However, in cobimetinib combined with vemurafenib-treated patients, both cell cycle and immune signature subgroups had comparable PFS. Cobimetinib combined with vemurafenib may abrogate the adverse impact of the cell-cycle signature. Clin Cancer Res; 23(17); 5238–45. ©2017 AACR.

Supplementary Data from JNJ-26481585, a Novel Second-Generation Oral Histone Deacetylase Inhibitor, Shows Broad-Spectrum Preclinical Antitumoral Activity

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Purpose: The association of tumor gene expression profiles with progression-free survival (PFS) outcomes in patients with BRAFV600-mutated melanoma treated with vemurafenib or cobimetinib combined with vemurafenib was evaluated. Experimental Design: Gene expression of archival tumor samples from patients in four trials (BRIM-2, BRIM-3, BRIM-7, and coBRIM) was evaluated. Genes significantly associated with PFS (P < 0.05) were identified by univariate Cox proportional hazards modeling, then subjected to unsupervised hierarchical clustering, principal component analysis, and recursive partitioning to develop optimized gene signatures. Results: Forty-six genes were identified as significantly associated with PFS in both BRIM-2 (n = 63) and the vemurafenib arm of BRIM-3 (n = 160). Two distinct signatures were identified: cell cycle and immune. Among vemurafenib-treated patients, the cell-cycle signature was associated with shortened PFS compared with the immune signature in the BRIM-2/BRIM-3 training set [hazard ratio (HR) 1.8; 95% confidence interval (CI), 1.3–2.6, P = 0.0001] and in the coBRIM validation set (n = 101; HR, 1.6; 95% CI, 1.0–2.5; P = 0.08). The adverse impact of the cell-cycle signature on PFS was not observed in patients treated with cobimetinib combined with vemurafenib (n = 99; HR, 1.1; 95% CI, 0.7–1.8; P = 0.66). Conclusions: In vemurafenib-treated patients, the cell-cycle gene signature was associated with shorter PFS. However, in cobimetinib combined with vemurafenib-treated patients, both cell cycle and immune signature subgroups had comparable PFS. Cobimetinib combined with vemurafenib may abrogate the adverse impact of the cell-cycle signature. Clin Cancer Res; 23(17); 5238–45. ©2017 AACR.

Slc17a5(-/-) mice represent an animal model for the infantile form of sialic acid storage disease (SASD). We analyzed genetic and histological time-course expression of myelin and oligodendrocyte (OL) lineage markers in different parts of the CNS, and related this to postnatal neurobehavioral development in these mice. Sialin-deficient mice display a distinct spatiotemporal pattern of sialic acid storage, CNS hypomyelination and leukoencephalopathy. Whereas few genes are differentially expressed in the perinatal stage (p0), microarray analysis revealed increased differential gene expression in later postnatal stages (p10-p18). This included progressive upregulation of neuroinflammatory genes, as well as continuous down-regulation of genes that encode myelin constituents and typical OL lineage markers. Age-related histopathological analysis indicates that initial myelination occurs normally in hindbrain regions, but progression to more frontal areas is affected in Slc17a5(-/-) mice. This course of progressive leukoencephalopathy and CNS hypomyelination delays neurobehavioral development in sialin-deficient mice. Slc17a5(-/-) mice successfully achieve early neurobehavioral milestones, but exhibit progressive delay of later-stage sensory and motor milestones. The present findings may contribute to further understanding of the processes of CNS myelination as well as help to develop therapeutic strategies for SASD and other myelination disorders. publisher: Elsevier articletitle: Progressive leukoencephalopathy impairs neurobehavioral development in sialin-deficient mice journaltitle: Experimental Neurology articlelink: http://dx.doi.org/10.1016/j.expneurol.2017.02.009 content_type: article copyright: © 2017 The Authors. Published by Elsevier Inc. ispartof: Experimental Neurology vol:291 pages:106-119 ispartof: location:United States status: published

Tumor angiogenesis is measured by counting microvessels in tissue sections at high power magnification as a potential prognostic or predictive biomarker. Until now, regions of interest1 1 ROI: region of interest. (ROIs) were selected by manual operations within a tumor by using a systematic uniform random sampling 2 SURS: systematic, uniform random sampling. (SURS) approach. Although SURS is the most reliable sampling method, it implies a high workload. However, SURS can be semi-automated and in this way contribute to the development of a validated quantification method for microvessel counting in the clinical setting. Here, we report a method to use semi-automated SURS for microvessel counting: • Whole slide imaging with Pannoramic SCAN (3DHISTECH) • Computer-assisted sampling in Pannoramic Viewer (3DHISTECH) extended by two self-written AutoHotkey applications (AutoTag and AutoSnap) • The use of digital grids in Photoshop® and Bridge® (Adobe Systems) This rapid procedure allows traceability essential for high throughput protein analysis of immunohistochemically stained tissue.

Bringing video images from camera to screen is a complex production process involving several intermediate steps and actors. The media sector is characterized by a high need for fast and reliable data transport, between production locations, post-production facilities, broadcasters and back-up locations. The growing size of video files and the stringent network requirements for live production have given rise to a combination of transport solutions including physical transport of raw materials, transport of proxies via best-effort Internet Protocol (IP) networks and transport of live video streams via Serial Digital Interface (SDI) over microwave, satellite, leased lines or optical fiber. Due to this fast-paced evolution, media companies face high investments costs for upgrading both end devices (e.g. cameras) and network transport infrastructure. As such, the cost of maintaining and upgrading a multitude of transport networks can be prohibitive. The MECaNO project Ill proposes a lossless packet switching technology developed that can guarantee delivery deadlines in order to create a single, lossless, IP-based production system which is realized by leveraging technological advances such as the decreasing cost of optical technology and the flow-based software defined networking (SDN) architecture. Apart from explaining the MECaNO solution, the goal of this paper is to quantify the economic impact of the proposed technological solution. The results show that the cost for the optical infrastructure (L1) remains relatively stable while the cost for L2-L3 technologies drops significantly leading to a total cost reduction of 27%.

Despite all efforts made to develop predictive biomarkers for antiangiogenic therapies, no unambiguous markers have been identified so far. This is due to among others the lack of standardized tests. This study presents an improved microvessel density quantification method in tumor tissue based on stereological principles and using whole-slide images. Vessels in tissue sections of different cancer types were stained for CD31 by an automated and validated immunohistochemical staining method. The stained slides were digitized with a digital slide scanner. Systematic, uniform, random sampling of the regions of interest on the whole-slide images was performed semi-automatically with the previously published applications AutoTag and AutoSnap. Subsequently, an unbiased counting grid was combined with the images generated with these scripts. Up to six independent observers counted microvessels in up to four cancer types: colorectal carcinoma, glioblastoma multiforme, ovarian carcinoma and renal cell carcinoma. At first, inter-observer variability was found to be unacceptable. However, after a series of consensus training sessions and interim statistical analysis, counting rules were modified and inter-observer concordance improved considerably. Every CD31-positive object was counted, with exclusion of suspected CD31-positive monocytes, macrophages and tumor cells. Furthermore, if interconnected, stained objects were considered a single vessel. Ten regions of interest were sufficient for accurate microvessel density measurements. Intra-observer and inter-observer variability were low (intraclass correlation coefficient > 0.7) if the observers were adequately trained.

Cutaneous squamous-cell carcinomas and keratoacanthomas are common findings in patients treated with BRAF inhibitors.We performed a molecular analysis to identify oncogenic mutations (HRAS, KRAS, NRAS, CDKN2A, and TP53) in the lesions from patients treated with the BRAF inhibitor vemurafenib. An analysis of an independent validation set and functional studies with BRAF inhibitors in the presence of the prevalent RAS mutation was also performed.Among 21 tumor samples, 13 had RAS mutations (12 in HRAS). In a validation set of 14 samples, 8 had RAS mutations (4 in HRAS). Thus, 60% (21 of 35) of the specimens harbored RAS mutations, the most prevalent being HRAS Q61L. Increased proliferation of HRAS Q61L-mutant cell lines exposed to vemurafenib was associated with mitogen-activated protein kinase (MAPK)-pathway signaling and activation of ERK-mediated transcription. In a mouse model of HRAS Q61L-mediated skin carcinogenesis, the vemurafenib analogue PLX4720 was not an initiator or a promoter of carcinogenesis but accelerated growth of the lesions harboring HRAS mutations, and this growth was blocked by concomitant treatment with a MEK inhibitor.Mutations in RAS, particularly HRAS, are frequent in cutaneous squamous-cell carcinomas and keratoacanthomas that develop in patients treated with vemurafenib. The molecular mechanism is consistent with the paradoxical activation of MAPK signaling and leads to accelerated growth of these lesions. (Funded by Hoffmann-La Roche and others; ClinicalTrials.gov numbers, NCT00405587, NCT00949702, NCT01001299, and NCT01006980.).

Purpose: Histone deacetylase (HDAC) inhibitors have shown promising clinical activity in the treatment of hematologic malignancies, but their activity in solid tumor indications has been limited. Most HDAC inhibitors in clinical development only transiently induce histone acetylation in tumor tissue. Here, we sought to identify a second-generation class I HDAC inhibitor with prolonged pharmacodynamic response in vivo, to assess whether this results in superior antitumoral efficacy. Experimental Design: To identify novel HDAC inhibitors with superior pharmacodynamic properties, we developed a preclinical in vivo tumor model, in which tumor cells have been engineered to express fluorescent protein dependent on HDAC1 inhibition, thereby allowing noninvasive real-time evaluation of the tumor response to HDAC inhibitors. Results: In vivo pharmacodynamic analysis of 140 potent pyrimidyl-hydroxamic acid analogues resulted in the identification of JNJ-26481585. Once daily oral administration of JNJ-26481585 induced continuous histone H3 acetylation. The prolonged pharmacodynamic response translated into complete tumor growth inhibition in Ras mutant HCT116 colon carcinoma xenografts, whereas 5-fluorouracil was less active. JNJ-26481585 also fully inhibited the growth of C170HM2 colorectal liver metastases, whereas again 5-fluorouracil/Leucovorin showed modest activity. Further characterization revealed that JNJ-26481585 is a pan-HDAC inhibitor with marked potency toward HDAC1 (IC50, 0.16 nmol/L). Conclusions: The potent antitumor activity as a single agent in preclinical models combined with its favorable pharmacodynamic profile makes JNJ-26481585 a promising second-generation HDAC inhibitor. The compound is currently in clinical studies, to evaluate its potential applicability in a broad spectrum of both solid and hematologic malignancies. (Clin Cancer Res 2009;15(22):684151)

Vesicular glutamate transporters 1 and 2 (VGLUT1, VGLUT2) show largely complementary distribution in the mature rodent brain and tend to segregate to synapses with different physiological properties. In the hippocampus, VGLUT1 is the dominate subtype in adult animals, whereas VGLUT2 is transiently expressed during early postnatal development. We generated and characterized VGLUT1 knockout mice in order to examine the functional contribution of this transporter to hippocampal synaptic plasticity and hippocampus-dependent spatial learning. Because complete deletion of VGLUT1 resulted in postnatal lethality, we used heterozygous animals for analysis. Here, we report that deletion of VGLUT1 resulted in impaired hippocampal long-term potentiation (LTP) in the CA1 region in vitro. In contrast, heterozygous VGLUT2 mice that were investigated for comparison did not show any changes in LTP. The reduced ability of VGLUT1-deficient mice to express LTP was accompanied by a specific deficit in spatial reversal learning in the water maze. Our data suggest a functional role of VGLUT1 in forms of hippocampal synaptic plasticity that are required to adapt and modify acquired spatial maps to external stimuli and changes.

R306465 is a novel hydroxamate-based histone deacetylase (HDAC) inhibitor with broad-spectrum antitumour activity against solid and haematological malignancies in preclinical models. R306465 was found to be a potent inhibitor of HDAC1 and -8 (class I) in vitro. It rapidly induced histone 3 (H3) acetylation and strongly upregulated expression of p21waf1,cip1, a downstream component of HDAC1 signalling, in A2780 ovarian carcinoma cells. R306465 showed class I HDAC isotype selectivity as evidenced by poor inhibition of HDAC6 (class IIb) confirmed by the absence of downregulation of Hsp90 chaperone c-raf protein expression and tubulin acetylation. This distinguished it from other HDAC inhibitors currently in clinical development that were either more potent towards HDAC6 (e.g. vorinostat) or had a broader HDAC inhibition spectrum (e.g. panobinostat). R306465 potently inhibited cell proliferation of all main solid tumour indications, including ovarian, lung, colon, breast and prostate cancer cell lines, with IC50 values ranging from 30 to 300 nM. Haematological cell lines, including acute lymphoblastic leukaemia, acute myeloid leukaemia, chronic lymphoblastic leukaemia, chronic myeloid leukaemia, lymphoma and myeloma, were potently inhibited at a similar concentration range. R306465 induced apoptosis and inhibited angiogenesis in cell-based assays and had potent oral in vivo antitumoral activity in xenograft models. Once-daily oral administration of R306465 at well-tolerated doses inhibited the growth of A2780 ovarian, H460 lung and HCT116 colon carcinomas in immunodeficient mice. The high activity of R306465 in cell-based assays and in vivo after oral administration makes R306465 a promising novel antitumoral agent with potential applicability in a broad spectrum of human malignancies.

Lithium has been the standard pharmacological treatment for bipolar disorder over the last 50 years; however, the molecular targets through which lithium exerts its therapeutic effects are still not defined. We characterized the phenotype of mice with a dysfunctional IMPA1 gene (IMPA1-/-) to study the in vivo physiological functions of IMPA1, in general, and more specifically its potential role as a molecular target in mediating lithium-dependent physiological effects. Homozygote IMPA1-/- mice died in utero between days 9.5 and 10.5 post coitum (p.c.) demonstrating the importance of IMPA1 in early embryonic development. Intriguingly, the embryonic lethality could be reversed by myo-inositol supplementation via the pregnant mothers. In brains of adult IMPA1-/- mice, IMPase activity levels were found to be reduced (up to 65% in hippocampus); however, inositol levels were not found to be altered. Behavioral analysis of the IMPA1-/- mice indicated an increased motor activity in both the open-field test and the forced-swim test as well as a strongly increased sensitivity to pilocarpine-induced seizures, the latter supporting the idea that IMPA1 represents a physiologically relevant target for lithium. In conclusion the IMPA1-/- mouse represents a novel model to study inositol homeostasis, and indicates that genetic inactivation of IMPA1 can mimic some actions of lithium.

To evaluate the involvement of frizzled receptors (Fzds) in oncogenesis, we investigated mRNA expression levels of several human Fzds in more than 30 different human tumor samples and their corresponding (matched) normal tissue samples, using real-time quantitative PCR. We observed that the mRNA level of Fzd5 was markedly increased in 8 of 11 renal carcinoma samples whilst Fzd8 mRNA was increased in 7 of 11 renal carcinoma samples. Western blot analysis of crude membrane fractions revealed that Fzd5 protein expression in the matched tumor/normal kidney samples correlated with the observed mRNA level. Wnt/beta-catenin signaling pathway activation was confirmed by the increased expression of a set of target genes. Using a kidney tumor tissue array, Fzd5 protein expression was investigated in a broader panel of kidney tumor samples. Fzd5 membrane staining was detected in 30% of clear cell carcinomas, and there was a strong correlation with nuclear cyclin D1 staining in the samples. Our data suggested that altered expression of certain members of the Fzd family, and their downstream targets, could provide alternative mechanisms leading to activation of the Wnt signaling pathway in renal carcinogenesis. Fzd family members may have a role as a biomarker. Copyright (C) 2004 S. Karger AG, Basel.

Background — In view of growing evidence of an important endothelial paracrine regulation of cardiac function, the present study investigated the role of cardiac endothelium-derived endothelin-1 (ET-1), prostaglandins, and nitric oxide (NO) during endotoxin-induced cardiomyopathy in rabbits. Methods and Results — Immunohistochemical studies showed a marked transient coinduction of the inducible isoforms of NO synthase (NOS-2) and cyclooxygenase (COX-2) in endocardial endothelium and coronary arteriolar endothelium of hearts 12 hours after intravenous administration of lipopolysaccharide (LPS+12h); staining for both isoforms was much weaker 24 hours later (LPS+36h). Nitrotyrosine localization was similar to that of NOS-2, suggesting a NOS-2–related endothelial formation of peroxynitrite in septic hearts. Contractile performance of papillary muscles was depressed in both LPS-treated groups. In the LPS+12h group, however, isometric twitches were significantly prolonged (482±14 versus 420±14 ms in the saline-treated group, P P Conclusions — Cardiac endothelial activation and myocardial sensitization to endothelium-derived mediators may be part of an adaptive response in the early (12 hours) stages of septic cardiomyopathy.

Abstract —Endocardial endothelium and endothelium of coronary vessels produce NO. Histochemical methods have suggested that coronary arterial endothelial cells contain more endothelial constitutive NO synthase (ecNOS) than does coronary venous endothelium. We have further investigated the distribution of ecNOS in cardiac endothelium using immunofluorescence and en face confocal microscopy of rat heart. In endocardial endothelium, confocal microscopy revealed distinct ecNOS labeling of peripheral cell borders, cytoplasmic labeling, and labeling of the Golgi complexes. Labeling of the cell borders and of the Golgi complexes was confirmed by double staining for ecNOS and for platelet and endothelial cell adhesion molecule or Golgi 58k protein, respectively. Cytoplasmic labeling was strongest in coronary arterial endothelium. The size of the ecNOS-labeled Golgi complexes decreased from coronary arterial endothelial cells (8.63±0.39 μm 2 , mean±SE of 5 rats) to endocardial endothelium (7.07±0.61 μm 2 ) and to coronary venous endothelium (3.65±0.20 μm 2 ). In addition, pixel intensity of ecNOS labeling was higher in arterial endothelial cells than in venous endothelial cells. Endothelium of myocardial capillaries also contained small ecNOS-labeled Golgi complexes. No correlation was observed between endothelial cell surface area and Golgi complex size. Caveolin-1 labeling was strongest in capillaries and did not coincide completely with ecNOS labeling in endocardial and venous endothelium. These results suggest that endocardial and coronary arterial endothelium in the rat have a higher synthetic activity and might express more ecNOS than is expressed by cardiac venous and capillary endothelium. The observed heterogeneity in ecNOS distribution might be related to the specific mechanochemical environment and function of each endothelial compartment.

Objectives: Based on in vitro studies, oxidized low-density lipoprotein (oxLDL) has been implicated in atherogenesis and the associated deficiency in endothelium-dependent relaxation. The aim of this study was to investigate the effects of in vivo exposure to oxLDL on intimal thickening and relaxing behaviour. Methods: Intimal thickening was evoked by the placement of silicone collars around the carotid arteries of the rabbit for 3 or 14 days. OxLDL (Cu2+-oxidized, 7 μg/h) or the vehicle phosphate-buffered saline (PBS) was infused in the collars via subdermally implanted osmotic minipumps. Results: The collared vessels receiving PBS developed discrete intimal thickening after 14 days (intima/media (I/M) ratio 11±2%). OxLDL infusion resulted in intimal thickening after 3 days and significantly enhanced the intimal thickness by 14 days (I/M ratio 98±16%). Collaring alone for 3 or 14 days and 3 days exposure to oxLDL did not impair the endothelium-dependent relaxations to acetylcholine or calcium ionophore, nor to the NO donors glyceryl trinitrate (GTN) and S -nitroso- N -acetylpenicillamine (SNAP). However, the sensitivity to acetylcholine was decreased after exposure to oxLDL for 14 days (−logEC50 oxLDL 6.95±0.11 vs. 7.52±0.11 collar alone) and the maximal relaxation to the endothelium-dependent agonist was reduced by 50%, this in the presence of a virtually intact endothelium. Complete relaxation was still obtained with the nitric oxide donors. Conclusion: Our results show for the first time that local vascular exposure to oxLDL in vivo promotes intimal thickening and inhibits endothelium-dependent dilation, thereby supporting an active role for oxLDL in the morphological and functional changes observed in atherosclerotic blood vessels.

Although the kidney generally has been regarded as an excellent source of carboxypeptidase M (CPM), little is known about its renal-specific expression level and distribution. This study provides a detailed localization of CPM in healthy and diseased human kidneys. The results indicate a broad distribution of CPM along the renal tubular structures in the healthy kidney. CPM was identified at the parietal epithelium beneath the Bowman’s basement membrane and in glomerular mesangial cells. Capillaries, podocytes, and most interstitial cells were CPM negative. Tumor cells of renal cell carcinoma subtypes lose CPM expression upon dedifferentiation. Tissue microarray analysis demonstrated a correlation between low CPM expression and tumor cell type. CPM staining was intense on phagocytotic tumor-associated macrophages. Immunoreactive CPM was also detected in the tumor-associated vasculature. The absence of CPM in normal renal blood vessels points toward a role for CPM in angiogenesis. Coexistence of CPM and the epidermal growth factor receptor (EGFR) was detected in papillary renal cell carcinoma. However, the different subcellular localization of CPM and EGFR argues against an interaction between these h proteins. The description of the distribution of CPM in human kidney forms the foundation for further study of the (patho)physiological activities of CPM in the kidney.

Like vascular endothelium, the EE plays a role in transendothelial transport, in coagulant and thrombotic processes, and in interactions with inflammatory cells. In addition, EE is involved in the modulation of cardiac performance of subjacent myocardium. EE dysfunction includes insufficient as well as excessive performance of any of its multiple functions. Dysfunction can progress from a disturbed modulation of myocardial performance and an imbalance in the release of growth factors to changes in EE cytoskeletal organization, with concomitant changes in transendothelial permeability, and in extreme cases, to loss of endothelial integrity and frank denudation. Structural and functional impairment of EE and of endocardial interstitial cells may be primary or secondary to the disease. Mechanical stress, various hormones and cytokines can initiate EE dysfunction. EE dysfunction may influence the development of cardiac failure in endo(myo)cardial fibrosis (Loeffler's endocarditis and carcinoid syndrome) and in dilated cardiomyopathy. Although Bouillaud, in 1836, was referring to endocarditis when stating: (quote: see text) his statement may presently find a much broader field of applicability in cardiology.

Interactions between the various cell types that make up the cardiovascular system are known to play an important role in maintaining homeostasis. One area about smooth muscle cells that has received little attention, despite the production of a wide variety of mediators by smooth muscle cells, is their effect on myocardial function. In this study, the myocardial contractile effects of four different types of dog aortic strips on rabbit papillary muscles were evaluated. Of these, medial vascular smooth muscle strips most consistently (65% of the time) produced a "vascular-derived contractile factor" (VDCF), which caused a 15% increase in isometric twitch tension and a 24% increase in isotonic twitch shortening with no change in twitch configuration. Endovascular strips with or without intact endothelium and complete aortic rings had less consistent effects. Vascular-derived contractile factor was stable after freezing at -80 degrees C, its activity was not modified by a broad spectrum peptidase, but it was heat-labile. The angiotensin II blocker, losartan, did not modify its effects. However, incubation with indomethacin did reduce, but did not eliminate, the contractile effects of vascular strips. The addition of alpha 1- and beta-blockers did not further modify the effects of VDCF. Endocardial endothelial removal increased the effects of VDCF. No correlation existed between endothelin levels and the contractile effects of vascular strips. It is concluded that VDCF is produced by the medial layer of large vessels but its exact cellular origin is uncertain. These findings expand the ever-increasing understanding of the inter-relationship between the structures that make up the cardiovascular system, and open the door to new studies evaluating the inter-relationship of vessels and myocardium.

Intimal thickening in human arteries is considered as a site of predilection for atherosclerosis. The placement of a flexible, physically nonconstrictive, silicone cuff around the rabbit carotid artery induced a neointima composed of smooth muscle cells (SMCs) within 14 days. To investigate possible alterations of the endothelial cells (ECs) during neointima formation, their morphology was examined with scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal microscopy. In the early postoperative period (6 hours), both cuffed and sham-operated arteries demonstrated small foci (5 to 200 microns) of denudation, presumably as a consequence of the manipulation. Within 24 hours the luminal surface of the cuffed and sham-operated arteries was completely covered with endothelium, which remained continuous throughout the study. However, after 1 week the ECs of the cuffed arteries contained a pronounced rough endoplasmic reticulum. From 6 hours until 3 days, polymorphonuclear leukocytes infiltrated the cuffed but not the sham-operated arteries from the lumen. Subendothelial SMC accumulation in the cuffed arteries began after this time period. At day 14 a full-blown neointima composed of longitudinally oriented SMCs had formed in the cuffed arteries. The sham-operated arteries did not develop a neointima. During neointima formation immunoreactivity for von Willebrand factor (vWf) increased in the ECs, and vWf was deposited in the extracellular spaces of the neointima. At day 14 the area of vWf deposits correlated positively with the area of the neointima (r = .73, P < .001). In subsequent weeks, the intimal area did not increase, and vWf deposits vanished from the neointimal matrix. The endothelium of the sham-operated arteries showed no change in vWf immunoreactivity compared with untreated arteries throughout the study. The altered ultrastructural morphology of the ECs and the concurrent vWf deposition in cuffed but not in sham-operated arteries point to alterations in EC function during the development of the neointima. The vWf secretion could possibly lead to increased adhesiveness of the extracellular matrix for the ECs as well as modulate neointima formation.

The heart wall with its complex trabecular structures is covered with a very thin layer of endocardial endothelial (EE) cells. EE cells appear early during cardiac development; they are involved in myocardial trabeculation and the formation of primitive nutrient vessels. This process precedes the development of coronary vessels, Purkinje fibers, and nerve fibers. EE has a different cell shape and cytoskeletal organization than vascular endothelium. The differences in permeability between EE and the coronary vascular endothelium in the subendocardial myocardium might assign characteristic electrochemical properties to the endocardium. Modulation of EE function by substances in the blood constitutes an important intracavitary autoregulation of muscle-pump performance of the heart, i.e., by altering the duration of contraction without significantly altering early contraction dynamics. Such an autoregulation resets the timing of ventricular relaxation and rapid filling with little effect on early systolic contraction and ejection. Both the release by the EE of various substances with inotropic properties and a trans-EE physicochemical control are postulated as possible underlying mechanisms.

Damage of endocardial endothelium by mechanochemical methods in isolated cardiac muscle induces typical changes in the contractile performance of the myocardium. Functional and morphological features of isolated cat papillary muscles treated with ultrasound, a new technique for in vitro damage of endocardial endothelium, were compared with damage by Triton X-100. Treatment with either short bursts of continuous wave ultrasound (25 W; 1.05 MHz) or with 1-s immersion in 0.5% Triton X-100 resulted in an irreversible abbreviation of twitch contraction with concomitant decrease in total peak twitch tension but without significant changes in maximal unloaded velocity of shortening. Decrease of total peak isometric tension was significantly more pronounced after Triton X-100 administration. Scanning electron microscopy showed an extracted endothelium following immersion in Triton X-100 and a nearly complete desquamated surface after ultrasound. Fluorochromes in muscles treated with Triton X-100 or ultrasound did not enter myocytes, which maintained a normal ultrastructure. After Triton X-100, more endocardial fibroblasts were labeled and showed ultrastructural damage than after ultrasound. Ultrasound constitutes a powerful technique for selective in vitro damage of endocardial endothelium. The technique is also suitable for experimental in vivo intracardiac application.

Persistent eosinophilia in humans is often associated with endocardial damage to the heart, but a causal relation has not been established. We investigated the effect of eosinophils and eosinophil supernatants obtained from eight hypereosinophilic patients on the contractile performance and endocardial morphology of isolated, electrically stimulated cat papillary muscle preparations (n = 16). All these eosinophil suspensions contained high proportions of "hypodense" or "activated" cells. Eosinophils (5-15 x 10(6) ml organ bath) or eosinophil culture supernatants (prepared by overnight incubation at 37 degrees C) when added to papillary muscles produced acute changes in contractile behavior of these muscles identical to the previously reported effects of selective endocardial damage: a reduction in time to peak isometric twitch tension causing a reduction in peak isometric tension but with no significant reduction in rate of tension development or in maximum unloaded shortening velocity. All of these muscle preparations showed severely damaged endocardium at scanning electron microscopy. Addition of eosinophils from hypereosinophilic patients to muscles with selectively damaged endocardium (by previous transient [1-second] exposure to 1% Triton X-100) produced no further change in contractile performance. No significant change in contractile performance or endocardial morphology of papillary muscles (n = 16) was observed after addition of eosinophils (7.5-10 x 10(6] or neutrophils (8-15 x 10(6] from normal subjects or of cell-free culture medium. Thus, activated human eosinophils produce specific morphological and functional changes suggestive of specific damage to endocardium of isolated feline cardiac muscle.

Although intense effort has been made in studying immune cell dynamics in tumors after treatment with targeted therapies and to recharacterize tumor stages based on their immune cell components, less is known about how baseline immune cell characteristics in tumors affect response to approved therapies. Here we describe baseline expression of immune regulators and CD8+ T-cell distribution in BRAF-mutated metastatic melanoma and their relationship with progression-free survival (PFS) on vemurafenib and chemotherapy. To our knowledge, we are the first to describe the baseline characteristics of tumor-infiltrating lymphocytes and their impact on outcomes in a large randomized, controlled trial in melanoma. Archival or baseline samples were collected from patients in 2 clinical trials (BRIM2 and BRIM3) and analyzed. A total of 252 RNA samples were prepared from formalin-fixed, paraffin-embedded (FFPE) tissue samples and profiled for expression using a panel of 96 immune genes on the Fluidigm platform (Fluidigm Corp). Additionally, for 277 patients from the BRIM3 trial, CD8+ T cells were detected by immunohistochemistry (IHC) and quantified using Definiens (Definiens AG). Patients were defined as high or low expressers based on a median cutoff, and hazard ratios (HRs) were determined by cox proportional hazards modeling of PFS. HRs refer to the comparison of high and low expressing groups, where an HR A discovery (BRIM2) – validation (BRIM3) scheme was applied to assess the prognostic value of the expression of immune-related genes. Samples from the BRIM3 dacarbazine and vemurafenib arms were compared to assess the predictive value of the markers on the treatment effect of vemurafenib. Of the genes tested, 26 met our discovery criteria in the BRIM2 trial and 2, IL7 and IL12A, were validated in BRIM3. In the vemurafenib arm of BRIM3, patients with high expression of IL7 had improved PFS compared with patients with low IL7, with an HR of 0.63 (95% confidence interval [CI]: 0.41-0.97; P=0.03); high expression of IL12A had an HR of 0.58 (0.38-0.88; P=0.01). Neither gene was associated with PFS in the dacarbazine-treated arm; however, high and low expressers of both genes benefited from vemurafenib treatment. Because immune contexture is known to be associated with outcomes, slides were stained for CD8+ T-cell content in 3 marker areas: center, peripheral, and invasive margin. In the dacarbazine arm, increased PFS was seen in patients with higher CD8+ T-cell content in the tumor center or periphery, with HRs of 0.58 (0.43-0.94; P=0.02) and 0.39 (0.25-0.59; P Based on these characterizations, tumor immune cell components seem to play important but varying roles in response to dacarbazine or vemurafenib treatment in BRAF-mutant melanoma. This abstract is also being presented as Poster B22. Citation Format: Matthew Wongchenko, Christina Rabe, Jeffrey Sosman, Grant McArthur, Yuanyuan Xiao, Houston Gilbert, Luc Andries, Mark Kockx, Hartmut Koeppen, Priti Hegde, Lukas Amler, Yibing Yan, Antoni Ribas. CD8+ T-cell distribution and immunomodulator expression in BRAF-mutant melanoma affect the response to BRAF inhibitor and chemotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr PR03.

Transmission electron microscopy (TEM) is currently the standard method to monitor autophagy in tissue. Because TEM is labor intensive, we recently questioned whether marker proteins could be found for unambiguous detection of autophagy in tissue using standard immunohistochemical techniques. Our findings indicated that the identification of autophagy-specific biomarkers for tissue is highly compromised due to lack of differential gene expression. In this respect, TEM remains an indispensable technique for evaluation of autophagy in situ. Nevertheless, immunohistochemical staining of microtubule-associated protein 1 light chain 3 (LC3) appeared to be a valuable technique to detect autophagosome formation in tissue but only when this protein is overexpressed, e.g., in GFP-LC3 transgenic animals. Furthermore, demonstration of granular cytoplasmic ubiquitin inclusions by immunohistochemistry may be an attractive technique to measure autophagic cell degeneration in some human pathologies such as neurodegenerative diseases, heart failure and atherosclerosis.

This poster discusses the performance of the TCP/IP stack and some common file transfer protocols when used in a scalable IP-based environment for digital media production. First, models were created to predict what to expect of these protocols. Afterwards, all protocols were extensively tested on a scalable setup, including clients, file servers (IBM xSeries), storage controllers and disks. Tuning the protocols, OS parameters and the use of specialized hardware (TCP Offload Engines) improved throughput and diminished processing power. Out of the models and current test results, a comparison was made between the different file transfer protocols, and the added value of an IP-based access network between user terminals and a central file server cluster is demonstrated. Up to 615 MB/s (5.16 Gbps) of traffic was reached from 1 quad Xeon IBM x366 (with attached storage) to a set of clients.

Autophagy is a regulated bulk degradation process involved in many different human pathologies. Transmission electron microscopy (TEM) is currently the only reliable method for monitoring autophagy in situ. Because TEM is labor intensive, we questioned whether useful marker proteins can be found for unambiguous detection of autophagy in tissue via routinely used colorimetric, immunohistochemical, or fluorescent techniques. Starved HepG2 hepatocytes and nutrient deprived liver tissue were used as a model for the initiation of autophagy. Our findings indicate that starvation-induced autophagy in HepG2 cells was associated neither with differential mRNA gene expression nor with changes in the expression level of known autophagy-related proteins. On the contrary, both transcription and translation were inhibited, suggesting that the identification of autophagy-specific biomarkers for tissue is highly compromised. Light chain 3 (LC3) protein, which is an attractive marker of autophagosomes, revealed a relatively low expression level in tissue and cultured cells, but could be detected via immunohistochemistry in liver from GFP-LC3 transgenic mice. The number of LC3 immunopositive dot-like structures was significantly upregulated in liver tissue from nutrient-deprived GFP-LC3 mice as compared with nonstarved control tissue. Our results suggest that LC3 immunostaining can be used as an alternative detection method for autophagy in situ, but only when this protein is overexpressed.

Circulating stem cells home within the myocardium, probably as the first step of a tissue regeneration process. This step requires adhesion to cardiac microvascular endothelium (CMVE). In this study, we studied mechanisms of adhesion between CMVE and mesenchymal stem cells (MSCs). Adhesion was studied in vitro and in vivo. Isolated 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled rat MSCs were allowed to adhere to cultured CMVE in static and dynamic conditions. Either CMVE or MSCs were pretreated with cytokines [IL-1beta, IL-3, IL-6, stem cell factor, stromal cell-derived factor-1, or TNF-alpha, 10 ng/ml]. Control or TNF-alpha-treated MSCs were injected intracavitarily in rat hearts in vivo. In baseline in vitro conditions, the number of MSCs that adhered to CMVE was highly dependent on the flow rate of the superfusing medium but remained significant at venous and capillary shear stress amplitudes. Activation of both CMVE and MSCs with TNF-alpha or IL-1beta before adhesion concentration dependently increased adhesion of MSCs at each studied level of shear stress. Consistently, in vivo, activation of MSCs with TNF-alpha before injection significantly enhanced cardiac homing of MSCs. TNF-alpha-induced adhesion could be completely blocked by pretreating either CMVE or MSCs with anti-VCAM-1 monoclonal antibodies but not by anti-ICAM-1 antibodies. Adhesion of circulating MSCs in the heart appears to be an endothelium-dependent process and is sensitive to modulation by activators of both MSCs and endothelium. Inflammation and the expression of VCAM-1 but not ICAM-1 on both cell types have a regulatory effect on MSC homing in the heart.

The Chk2 kinase is a tumor suppressor and key component of the DNA damage checkpoint response that encompasses cell cycle arrest, apoptosis, and DNA repair. It has also been shown to have a role in replicative senescence resulting from dysfunctional telomeres. Some of these functions are at least partially exerted through activation of the p53 transcription factor. High-level expression of virally transduced Chk2 in A549 human lung carcinoma cells led to arrested proliferation, apoptosis, and senescence. These were accompanied by various molecular events, including p21Waf1/Cip1 (p21) transcriptional induction, consistent with p53 activation. However, Chk2-dependent senescence and p21 transcriptional induction also occurred in p53-defective SK-BR-3 (breast carcinoma) and HaCaT (immortalized keratinocyte) cells. Small interfering RNA–mediated knockdown of p21 in p53-defective cells expressing Chk2 resulted in a decrease in senescent cells. These results revealed a p53-independent role for Chk2 in p21 induction and senescence that may contribute to tumor suppression and genotoxic treatment outcome.

Phagocyte recognition of cells undergoing apoptosis is a rapid, efficient way of removing unwanted cells from tissue. The uptake of apoptotic cells prevents the release of potentially toxic cell contents that might otherwise damage neighbouring cells and elicit an inflammatory response. The aim of this work was to evaluate a simple cell culture assay to study phagocytosis of cells undergoing apoptosis. Fluorescent negatively charged beads (1 microm) or fluorescently labelled apoptotic cells, derived from etoposide-treated human monocytes (U937), were co-incubated with J774 cells or human peripheral blood macrophages for 1 h. Flow cytometry (FCM) showed an efficient uptake of both beads and apoptotic bodies. Phagocytosis of apoptotic cells but not of beads was significantly inhibited when macrophages were pre-incubated with cytochalasin D, suggesting that an experimental system based on beads is not an appropriate model of phagocytosis of apoptotic cells.

The naturally occurring cyclic tetrapeptide chlamydocin is a very potent inhibitor of cell proliferation. Here we show that chlamydocin is a highly potent histone deacetylase (HDAC) inhibitor, inhibiting HDAC activity in vitro with an IC50 of 1.3 nM. Like other HDAC inhibitors, chlamydocin induces the accumulation of hyperacetylated histones H3 and H4 in A2780 ovarian cancer cells, increases the expression of p21(cip1/waf1), and causes an accumulation of cells in G(2)/M phase of the cell cycle. In addition, chlamydocin induces apoptosis by activating caspase-3, which in turn leads to the cleavage of p21(cip1/waf1) into a 15-kDa breakdown product and drives cells from growth arrest into apoptosis. Concomitant with the activation of caspase-3 and cleavage of p21(cip1/waf1), chlamydocin decreases the protein level of survivin, a member of the inhibitor of apoptosis protein family that is selectively expressed in tumors. Although our data indicate a potential link between degradation of survivin and activation of the apoptotic pathway induced by HDAC inhibitors, stable overexpression of survivin does not suppress the activation of caspase-3 or cleavage of p21(cip1/waf1) induced by chlamydocin treatment. The decrease of survivin protein level is mediated by degradation via proteasomes since it can be inhibited by specific proteasome inhibitors. Taken together, our results show that induction of apoptosis by chlamydocin involves caspase- dependent cleavage of p21(cip1/waf1), which is strikingly associated with proteasome-mediated degradation of survivin.

Background —Endocardial (EE) and myocardial capillary vascular endothelial (myocap VE) cells have been shown to modulate the contractile characteristics of myocardium in a calcium-dependent manner. We evaluated the endothelial-myocardial interaction in the rat postinfarction myocardial infarction (MI) model and the effects of captopril. Methods and Results —Wistar rats were divided into 4 groups treated for 4 weeks: (1) control; (2) infarcted controls (left anterior coronary artery ligation); (3) infarcted+captopril 2 g/L in drinking water; and (4) infarct+captopril+triton intracoronary injection. Coronary VE function was evaluated by infusion of serotonin in Langendorff preparations (n=31), and the myocardial contractile characteristics were investigated by use of isolated papillary muscles (n=44). Cardiac mRNA for endothelial constitutive nitric oxide synthase (ecNOS) was measured, and its cellular location was evaluated by immunohistochemistry. Serotonin-induced increase in coronary flow was decreased in infarct controls compared with controls (4.6% versus 53.4%, P 2 ). Cardiac ecNOS mRNA decreased in the control infarct group but remained normal in the infarct+captopril group. Conclusions —Chronic postinfarction endothelium-induced coronary vasodilatation is impaired, and both EE and myocap VE dysfunction contribute to myocardial depression. Captopril use prevents these abnormalities and the reduction of cardiac ecNOS mRNA.

Abstract —In this study, the involvement of polymorphonuclear leukocytes (PMNs) in the development of intimal thickening was investigated. A fibromuscular intima was induced by placing a silicone collar around the rabbit carotid artery for 3 days or 2 weeks; the contralateral artery was sham operated. Rabbits received placebo treatments (groups 1 and 3), granulocyte-colony stimulating factor (group 2; G-CSF, 20 μg · kg −1 · d −1 , delivered by subcutaneous osmotic pumps), or an anti-CD18 monoclonal antibody (group 4; 1.5 mg/kg IV). The G-CSF treatment raised the peripheral PMN count 5- to 12-fold but had no effect on intimal thickening on day 3, 12, or 14. A single injection of anti-CD18 prevented PMN extravasation 6 hours after collar implantation without influencing intimal hyperplasia on day 14. Repeated daily administration of anti-CD18 strongly bound to CD18 on peripheral PMNs and inhibited both PMN-dependent plasma extravasation in the skin and accumulation of CD14-immunoreactive leukocytes in the intima and media. However, anti-CD18 did not suppress early intimal thickening or accumulation of α-smooth muscle actin–immunoreactive cells by day 3. It thus appears that the PMN influx in the intima and media evoked by the perivascular collar is of little functional relevance to the subsequent smooth muscle cell migration and intimal thickening in this model.

Endocardial endothelium and vascular endothelium of myocardial capillaries share common features as modulators of cardiac performance, rhythmicity and growth. Growing evidence suggests differences between these two cardiac endothelial cell types with regard to developmental, morphological and functional properties. A major difference probably resides in the way and extent by which these endothelial cells perceive and transmit signals.

Abstract Oxidized LDL (oxLDL) has been implicated in atherogenesis on the basis of in vitro studies and is present in atherosclerotic lesions. The aim of this study was to investigate the effects of LDL and oxLDL on intimal thickening in vivo. Intimal thickening was evoked by the placement of silicone collars around the carotid arteries of rabbits for 2 weeks. The collars were connected to osmotic minipumps containing LDL (7 μg h −1 , n=16 arteries), oxLDL (Cu 2+ oxidized, 7 μg h −1 , n=16), or phosphate-buffered saline (5 μL h −1 , n=16). Segments proximal to the collars served as controls. Collar placement without lipoprotein application resulted in the appearance of α-SMC actin–immunoreactive cells in the intima, thereby increasing the intimal thickness from 5±1 to 26±5 μm. The perivascular infusion of LDL or oxLDL within the collar significantly enhanced the development of the intima ninefold and sevenfold, respectively. The large intimas resulting from lipoprotein exposure were infiltrated by macrophages and T lymphocytes, and the intimal collagen area was increased from 5±2% in the discrete collar-induced intima to ≈20% in the lipoprotein-evoked lesions. In conclusion, the local vascular application of LDL, oxidized in vitro or possibly in vivo, elicited an inflammatory-fibroproliferative response characteristic of arteriosclerotic lesions, thereby demonstrating an active role for this class of lipoproteins in the disease process.

Endothelial integrity and function may be an important determinant for long-term success of allograft heart valves. To determine the optimal storage temperatures for preserving long-term endothelial function of porcine aortic valves, different storage temperatures and different periods of time were investigated. Fresh valves were either a) stored at 4 °C with or without 10% fetal calf serum (FCS) supplement, for 1, 2,4, 7, 14, 21, and 28 days. b) cryopreserved for 2, 4, and 8 weeks without FCS in either at − 80 °C or at − 170 °C. c) cryopreserved in long-term storage (as long as 1 year), with or without FCS, at − 170 °C. Viability of endothelial cells was assessed through measurement of the production of prostacyclin (PGI2) in basal and bradykinin (BK) stimulated conditions, during in vitro incubation of the valve cusps at 37 °C, using a radioimmunoassay for 6-oxo-prostaglandin F1α (PGF1α). Endothelial morphological variations in valves stored at 4 °C were evaluated by scanning electron microscopy (SEM), in valves cryopreserved at − 80 °C and − 170 °C were evaluated by confocal scanning laser microscopy (CSLM). Results: a) With storage at 4 °C, after 4 days the valves produced already significantly less (p < 0.05) PGI2 than fresh preparations in both basal (0.21 ± 0.04 vs 3.56 ± 0.03 ng/ml · cm2) and stimulated conditions (4.17 ± 0.36 vs 24.23 ± 1.83). Morphological changes could not yet be distinguished with SEM at that time. When the storage period was extended, the levels of PGI2 further diminished; after 14 days, PGI2 release could no longer be detected. b) In cryopreserved valves, PGI2 production was similar for as long as 2 weeks of storage either at − 80 °C or at − 170 °C in basal (2.69 ± 0.63 vs 2.93 ± 0.51) and stimulated (16.43 ± 3.19 vs 16.50 ± 2.57) conditions. After 8 weeks, no PGI2 release could be detected in valves stored at − 80 °C. c) After 6 months storage at − 170 °C, the PGI2 production was significantly (p < 0.05) reduced compared with fresh valves; it then remained constant for as long as 1 year. The valves stored with fetal calf serum produced significantly (p < 0.05) less PGI2 than did those without FCS. CLSM showed that large areas of valve cusps retain intact endothelial cells after 1 year stored at − 170 °C. For longer cryopreserved banking, we recommend storing heart valves at − 170 °C instead of at − 80 °C for maintaining viability of endothelial cells. Fetal calf serum would harm endothelial viability during long-term cryopreservation.

In 1986, Brutsaert et al. observed that the endocardial endothelium (EE) directly controls performance of the myocardium. From this observation, the existence of an endocardium-mediated intracavitary autoregulation of cardiac performance has been postulated. Recent discoveries on morphology and function of the endocardium have substantiated this hypothesis. Morphologically, phenotypic expression of several receptors, tight and gap junctions, adhesion molecules, intercellular clefts, and a specific organization of the cytoskeleton distinguishes the EE from other endothelial subtypes and emphasizes a structural and functional specialization. Functionally, the EE imparts a twitch-prolonging and positive inotropic effect on cardiac muscle, apparently by enhancing the responsiveness of myocardial contractile proteins to Ca2+. EE-mediated control of myocardial performance resembles length/volume-mediated control and interacts with neurohumoral-mediated control. EE cells respond to physical and humoral stimuli with release of several substances that have important inotropic actions on the myocardium. Specific electrophysiological properties determine the intracellular Ca2+ increase in response to external stimuli and modulate the release of these substances. An asymmetrical distribution of ion channels between the luminal and abluminal membrane of the EE suggests a specific transendothelial transport of ions over the endocardium. Accordingly, the endocardium is highly specialized for communication with and regulation of the myocardium. It will be interesting to evaluate endocardium-mediated control of cardiac performance in pathophysiological conditions.

Cardiac endothelial cells, regardless of whether they are from endocardial or from coronary (micro)vascular origin, directly modulate performance of the subjacent cardiomyocytes, resulting in control of the onset of ventricular relaxation and rapid filling of the heart. This review summarizes major features of the morphology, embryology, and comparative physiology of cardiac endothelial cells as well as the experimental observations on how cardiac endothelial cells affect the mechanical performance of the heart. As for the underlying mechanisms of the interaction between cardiac endothelial cells and cardiomyocytes, two working hypotheses have been postulated over the past years; (1) interaction mediated through a trans-endothelial physicochemical gradient for various ions (active blood-heart barrier), and (2) interaction mediated through the release by the cardiac endothelial cells of various cardioactive substances, eg, nitric oxide, endothelin, and prostacyclin. These two mechanisms may act in concert or in parallel.

Abstract Cardiac endothelium, both coronary and endocardial, produces a number of inotropic molecules. Changes in cardiac endothelial function by substances in the superfusing blood may thus participate in the control of muscle-pump performance of the heart. Reactive oxygen species (ROS) have been implicated in normal and pathological vascular physiology by influencing vascular endothelial function. Therefore, we examined the influence of ROS on endocardial endothelial modulation of myocardial performance. Right ventricular cat papillary muscles were briefly (15 s) exposed to electrolysis-generated ROS. Peak total isometric twitch tension and peak rate of tension development increased by 7.8±0.7% ( P P P N G -nitro- l -arginine methyl ester and indomethacin (n=5) or with atenolol (n=6) did not influence the inotropic effect. Confocal scanning laser microscopic observations of muscles stained with viability tracers (n=9) revealed that significantly more but not all endocardial endothelial cells were damaged in electrolysis-treated muscles than in control muscles (42±5% versus 14±4%, P

The spreading behaviour of dissociated hypoblast cells on and besides a band of aligned fibrils associated with the basal lamina of the epiblast was investigated by the use of scanning electron microscopy. A horse-shaped band of aligned fibrils, first demonstrated by Wakely and England (1979), is present during the gastrulation stages of chicken embryos on the ventral side of the epiblast at the cranial and lateral borders of the area pellucida. The basal lamina of the area pellucida situated inside the fibrillar band enables the spreading and probably the locomotion of dissociated cells, which appeared as polarized cells. Numerous cells were also found on the fibrillar band, and these cells lacked distinct lamellae and a polarized shape. Extensions of the cells contacted the extracellular fibrils and, at these sites of contact, the pattern of the fibrils was frequently deformed. From these observations and from previous results emerged the concept that spreading and locomotion of dissociated hypoblast cells, as well as single mesoblast cells and healing hypoblast epithelium, are inhibited by the band of extracellular fibrils, which acts as a physical barrier. The cell biological basis of the mechanism by which extracellular fibrils associated with the basal lamina arrest the migration of hypoblast and mesoblast cells, but guide the migration of primordial germ cells, is discussed.