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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.

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.

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.

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.

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.

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 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.

The aim of this study was to compare different techniques of aortic valve cryopreservation by studying the viability of the endothelial cells. Viability was assessed by measuring their in vitro prostacyclin (PGI2) production under basal and stimulated conditions. Fresh and cryopreserved porcine valves were incubated at 37 degrees C in tissue culture medium and PGI2 content in the medium was measured every 15 min up to 300 min. Cryopreservation by the older procedure A included 5% fetal calf serum (FCS) in the preservation medium, a plastic box inside a freezing plastic bag, a cooling schedule approximating -2 degrees C/min, a long thawing time and few dilution steps of the cryoprotectant dimethylsulphoxide (DMSO). The newer procedure B differed from A in packaging, freezing and thawing rates and DMSO dilution. Procedure C was similar to B with the exception that FCS was omitted. Leaflets preserved by procedure A produced significantly less prostacyclin as compared to those treated according to procedures B or C. We conclude that minor differences in the cryopreservation method can become critical to endothelial functional viability.

Irradiation of isolated cardiac muscle by high-power, high-frequency, continuous wave ultrasound selectively damages endocardial endothelium (EE). We evaluated this ultrasound effect in vivo on the performance of the intact ejecting canine left ventricle (LV). A cylindrical ultrasound probe (0.9 MHz, 25 W), mounted on a catheter, was inserted in the LV cavity through an apical stab wound and was activated for 60, 120, and 240 s, followed each time by a recovery period of 10–15 min. Ultrasound transiently and repeatedly abbreviated the time interval from end diastole to peak (-)dP/dt (from 241 ± 30 to 229 ± 32 ms after 240 s; P < 0.001), accelerated LV pressure fall, did not alter peak (+)-dP/dt or peak systolic pressure, increased diastolic and systolic segment lengths, and decreased fractional shortening. Microscopic analysis revealed dispersed granulocytes attached to the EE. EE cells were visibly damaged only in a limited area surrounding the probe. Accordingly, high-power, high-frequency, continuous wave ultrasound reversibly modulated LV performance, presumably by transient alteration of EE function. cardiac function; contractility; diastole; relaxation Submitted on May 7, 1991 Accepted on April 28, 1992