scholarly journals Beta actin and its mRNA are localized at the plasma membrane and the regions of moving cytoplasm during the cellular response to injury.

1991 ◽  
Vol 112 (4) ◽  
pp. 653-664 ◽  
Author(s):  
T C Hoock ◽  
P M Newcomb ◽  
I M Herman
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
In Situ Hybridization ◽  
Cellular Response ◽  
Stress Fibers ◽  
Wound Edge ◽  
Actin Isoforms ◽  
Beta Actin ◽  
Cell Biol

Previous work in our laboratory has shown that microvascular pericytes sort muscle and nonmuscle actin isoforms into discrete cytoplasmic domains (Herman, I. M., and P. A. D'Amore. 1985. J. Cell Biol. 101:43-52; DeNofrio, D.T.C. Hoock, and I. M. Herman. J. Cell. Biol. 109:191-202). Specifically, muscle (alpha-smooth) actin is present on the stress fibers while nonmuscle actins (beta and gamma) are located on stress fibers and in regions of moving cytoplasm (e.g., ruffles, lamellae). To determine the form and function of beta actin in microvascular pericytes and endothelial cells recovering from injury, we prepared isoform-specific antibodies and cDNA probes for immunolocalization, Western and Northern blotting, as well as in situ hybridization. Anti-beta actin IgG was prepared by adsorption and release of beta actin-specific IgG from electrophoretically purified pericyte beta actin bound to nitrocellulose paper. Anti-beta actin IgGs prepared by this affinity selection procedure showed exclusive binding to beta actin present in crude cell lysates containing all three actin isoforms. For controls, we localized beta actin as a bright rim of staining beneath the erythrocyte plasma membrane. Anti-beta actin IgG, absorbed with beta actin bound to nitrocellulose, failed to stain erythrocytes. Simultaneous localization of beta actin with the entire F-actin pool was performed on microvascular pericytes or endothelial cells and 3T3 fibroblasts recovering from injury using anti-beta actin IgG in combination with fluorescent phalloidin. Results of these experiments revealed that pericyte beta actin is localized beneath the plasma membrane in association with filopods, pseudopods, and fan lamellae. Additionally, we observed bright focal fluorescence within fan lamellae and in association with the ends of stress fibers that are preferentially associated with the ventral plasmalemma. Whereas fluorescent phalloidin staining along the stress fibers is continuous, anti-beta actin IgG localization is discontinuous. When injured endothelial and 3T3 cells were stained through wound closure, we localized beta actin only in motile cytoplasm at the wound edge. Staining disappeared as cells became quiescent upon monolayer restoration. Appearance of beta actin at the wound edge correlated with a two- to threefold increase in steady-state levels of beta actin mRNA, which rose within 15-60 min after injury and returned to noninjury levels during monolayer restoration. In situ hybridization revealed that transcripts encoding beta actin were localized at the wound edge in association with the repositioned protein. Results of these experiments indicate that beta actin and its encoded mRNA are polarized at the membrane-cytoskeletal interface within regions of moving cytoplasm.

scholarly journals Microvascular pericytes contain muscle and nonmuscle actins.

1985 ◽  
Vol 101 (1) ◽  
pp. 43-52 ◽  
Author(s):  
I M Herman ◽  
P A D'Amore
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Plasma Membrane ◽  
Fluorescence Microscopy ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Stress Fibers ◽  
Fiber Bundles

We have affinity-fractionated rabbit antiactin immunoglobulins (IgG) into classes that bind preferentially to either muscle or nonmuscle actins. The pools of muscle- and nonmuscle-specific actin antibodies were used in conjunction with fluorescence microscopy to characterize the actin in vascular pericytes, endothelial cells (EC), and smooth muscle cells (SMC) in vitro and in situ. Nonmuscle-specific antiactin IgG stained the stress fibers of cultured EC and pericytes but did not stain the stress fibers of cultured SMC, although the cortical cytoplasm associated with the plasma membrane of SMC did react with nonmuscle-specific antiactin. Whereas the muscle-specific antiactin IgG failed to stain EC stress fibers and only faintly stained their cortical cytoplasm, these antibodies reacted strongly with the fiber bundles of cultured SMC and pericytes. Similar results were obtained in situ. The muscle-specific antiactin reacted strongly with the vascular SMC of arteries and arterioles as well as with the perivascular cells (pericytes) associated with capillaries and post-capillary venules. The non-muscle-specific antiactin stained the endothelium and the pericytes but did not react with SMC. These findings indicate that pericytes in culture and in situ possess both muscle and nonmuscle isoactins and support the hypothesis that the pericyte may represent the capillary and venular correlate of the SMC.

Actin mRNA isoforms are differentially sorted in normal osteoblasts and sorting is altered in osteoblasts from a skeletal mutation in the rat

1998 ◽  
Vol 111 (9) ◽  
pp. 1287-1292 ◽  
Author(s):  
H. Watanabe ◽  
E.H. Kislauskis ◽  
C.A. Mackay ◽  
A. Mason-Savas ◽  
S.C. Marks
Keyword(s):  
In Situ Hybridization ◽  
Bone Resorption ◽  
Cell Types ◽  
Mrna Isoforms ◽  
Actin Isoforms ◽  
Beta Actin ◽  
Cell Processes ◽  
Actin Mrna ◽  
Actin Isoform

Actin isoform sorting has been shown to occur in a variety of cell types in culture. To this list we add osteoblasts, in which we show by in situ hybridization that beta-actin is distributed primarily in cell processes and on one side of the nucleus and gamma-actin has a perinuclear distribution. Osteoblasts from the skeletal mutation toothless (tl), evaluated under identical conditions, fail to sort these actin isoforms differentially and exhibit diffuse labeling as their major manifestation. Northern analyses of actin mRNAs showed no differences between normal and mutant cultures. Shortened osteoblast life span and an inability to direct osteoclast-mediated bone resorption have recently been demonstrated in tl mutants. The present results suggest that a failure of osteoblasts to sort actin mRNAs may be related to one or both of these pathological manifestations in this mutation and represent, to our knowledge, the first correlation of an actin mRNA-sorting abnormality with a mammalian disease.

Comparative Aspects Of The Occluding Junctions In The Pulmonary And Bronchial Microcirculation

1981 ◽  
Author(s):  
H Bartels
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Freeze Fracture ◽  
Continuous System ◽  
Intramembranous Particles ◽  
Vasoactive Substances ◽  
Cell Biol ◽  
Occluding Junctions ◽  
Pulmonary Microcirculation

Compared with the pulmonary microcirculation the bronchial microcirculation reacts with a large increase in permeability after administration of vasoactive substances (Pietra et al., Cire. Res. 29, 323, 1971). Thus the occluding junctions between the endothelial cells of the rat pulmonary and bronchial microcirculation were investigated by means of freeze-fracture replicas in order to establish differences in their substructure which could be responsible for the different permeability properties. The prevailing feature of the occluding junctions between the endothelial cells of the pulmonary microvascular bed was a continuous system of anastomosing membrane foldings,carrying rows of intramembranous particles, which were usually located on face E of the split plasma membrane. This type of occluding junction is characteristic of capillaries (Simionescu et al., J. Cell Biol. 67, 863, 1975) . In the bronchial microcirculation many of the endothelial occluding junctions showed discontinuous assemblies of membrane foldings, which were lacking particles on face E. This type of “open” junction is typically localized at the venular end of the microvascular bed and susceptible to vasoactive substances (Simionescu et al., J. Cell Biol. 78, 27, 1978). It is suggested that the different permeability properties of the pulmonary and bronchial microcirculation is due to the large amount of postcapillary venules with their morphologically and functionally characteristic occluding junctions.

scholarly journals Simultaneous in situ detection of two mRNAs in the same cell using riboprobes labeled with biotin and 35S.

1990 ◽  
Vol 38 (7) ◽  
pp. 917-922 ◽  
Author(s):  
S Ozden ◽  
C Aubert ◽  
D Gonzalez-Dunia ◽  
M Brahic
Keyword(s):  
In Situ Hybridization ◽  
Theiler's Virus ◽  
Bhk Cells ◽  
Beta Actin ◽  
Actin Mrna ◽  
High Level ◽  
Radiolabeled Probe ◽  
In Situ Detection

We used 35S-labeled and biotinylated cRNAs (riboprobes) to detect simultaneously two different mRNAs by in situ hybridization. In a first step we established the conditions under which each type of probe achieved the same high level of sensitivity. We then used these conditions to hybridize BHK cells infected with Theiler's virus, a murine picornavirus, with a mixture of a virus-specific biotinylated riboprobe and a 35S-labeled riboprobe specific for beta-actin mRNA. Both mRNAs could be detected in the same cell, although the sensitivity achieved by the radiolabeled probe was reduced by about 40% by the simultaneous hybridization with the biotinylated probe.

Involvement of Erythropoietin Expression in Acupuncture Preconditioning-Induced Ischemic Tolerance

2012 ◽  
Vol 554-556 ◽  
pp. 1650-1655 ◽  
Author(s):  
Xue Mei Han ◽  
Hong Tao Wei ◽  
Song Yan Liu
Keyword(s):  
Endothelial Cells ◽  
In Situ Hybridization ◽  
Cerebral Ischemia ◽  
Vascular Endothelial Cells ◽  
Focal Cerebral Ischemia ◽  
Peripheral Zone ◽  
Vascular Endothelial ◽  
Neuroprotective Effects ◽  
Protein Expressions

Abstract Objective To investigate the expression of erythropoietin (EPO) after acupuncture preconditioning plus focal cerebral ischemia treatment. Methods Rat focal cerebral ischemia model and acupuncture preconditioning model were established. Animals were randomly assigned into different groups: control (focal cerebral ischemia) and acupuncture preconditioning plus focal cerebral ischemia, with 8 rats for each group. The expression of EPO after different treatments was determined by histological examination, immunohistochemistry and in situ hybridization. Results The mRNA and protein expressions of EPO could be detected in survival and necrotic neurons, glia as well as vascular endothelial cells. Focal cerebral ischemia promoted the expression of EPO. Significant enhanced EPO level was found in the ischemic peripheral zone after acupuncture preconditioning (P < 0.05). Conclusion Our results demonstrated that acupuncture preconditioning enhanced the expression of EPO in neurons, glia and vascular endothelial cells the ischemic peripheral zone, suggesting the involvement of EPO in acupuncture preconditioning-induced neuroprotection following focal cerebral ischemia. EPO may exert neuroprotective effects through promoting neurotrophic support and angiogenesis.

Horizontal Transfer of DNA by the Uptake of Apoptotic Bodies

Blood ◽  
1999 ◽  
Vol 93 (11) ◽  
pp. 3956-3963 ◽  
Author(s):  
Lars Holmgren ◽  
Anna Szeles ◽  
Eva Rajnavölgyi ◽  
Judah Folkman ◽  
Georg Klein ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
In Situ Hybridization ◽  
Expression Pattern ◽  
Cell Lines ◽  
Immunofluorescent Staining ◽  
Apoptotic Bodies ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Aortic Endothelial

In this study we have raised the question of whether DNA can be transferred from one cell to another by phagocytosis of apoptotic bodies. We have used integrated copies of the Epstein-Barr virus (EBV) as a marker to follow the fate and expression pattern of apoptotic DNA in the phagocytotic host. Apoptosis was induced in EBV-carrying cell lines by irradiation before cultivation with either human fibroblasts, macrophages, or bovine aortic endothelial cells. Analysis of the expression pattern of EBV-encoded genes was performed by immunofluorescent staining as well as in situ hybridization. Cocultivation of apoptotic bodies from lymphoid cell lines containing integrated but not episomal copies of EBV resulted in expression of the EBV-encoded genes EBER and EBNA1 in the recipient cells at a high frequency. Fluorescence in situ hybridization analysis showed uptake of human chromatin as well as integrated EBV-DNA into the nuclei of bovine aortic endothelial cells. These data show that DNA may be rescued and reused from apoptotic bodies by somatic cells. In addition, our findings suggest that apoptotic bodies derived from EBV-carrying B lymphocytes may serve as the source of viral transfer to cells that lack receptors for the EBV virus in vivo.

scholarly journals The expression of renin and the formation of angiotensin II in bovine aortic endothelial cells

2000 ◽  
Vol 164 (2) ◽  
pp. 207-214 ◽  
Author(s):  
F Xiao ◽  
GP Vinson ◽  
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
In Situ Hybridization ◽  
Ang Ii ◽  
Rt Pcr ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Human Renin ◽  
Aortic Endothelial

One controversy in the field of vascular angiotensin generation has surrounded the nature and particularly the source of vascular renin. This study investigated the expression of renin protein and its mRNA in aortic endothelial cells using immunocytochemistry, Western blotting, in situ hybridization and reverse transcription PCR (RT-PCR). Using a monoclonal antibody against human renin, immunocytochemical analysis revealed positive immunoreactivity in the cytoplasm of cultured bovine aortic endothelial cells. Immunoblotting of solubilized proteins separated by SDS-PAGE from cultured aortic endothelial cells identified two immunoreactive species with molecular masses of approximately 37-40 kDa. In situ hybridization showed that renin mRNA was localized in the cytoplasm of these cells. Using RT-PCR of RNA extracted from bovine aortic endothelial cells with primers specific for human renin, a clear single band was detected, which had the predicted size of 142 bp for (pro)renin. Angiotensin II (Ang II) was assayed in conditioned medium (CM) from cultured bovine aortic endothelial cells, and in addition, the effects of Ang II and CM on the proliferation of aorta smooth muscle cells (ASMC) were also studied. The results showed that CM contained Ang II equivalent to 15.05+/-4.67 pg/10(6) cells. Assay of smooth muscle cell proliferation by cell number, and by tritiated thymidine uptake, showed that proliferative responses in the presence of Ang II at a concentration of 10(-6)M were evident within 1 day of subculture, and cell numbers were nearly twice those of controls after 2 days. Thymidine incorporation into ASMC was also increased by Ang II in a dose-dependent manner and by endothelial cell CM. In both cases, stimulated proliferation was inhibited by the Ang II type 1 (AT1) receptor selective antagonist, losartan. These findings suggest that these vascular endothelial cells are a source of locally synthesized renin that may thus be involved in vascular Ang II generation. They also suggest that Ang II produced by the endothelial cells may be secreted and stimulate ASMC proliferation via the AT1 receptor.

Presence and distribution of endothelin-1 gene expression in human kidney

1994 ◽  
Vol 267 (4) ◽  
pp. F679-F687 ◽  
Author(s):  
C. Pupilli ◽  
M. Brunori ◽  
N. Misciglia ◽  
C. Selli ◽  
L. Ianni ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
In Situ Hybridization ◽  
Renal Cortex ◽  
Cdna Probe ◽  
Renal Medulla ◽  
Human Kidney ◽  
Renal Tumors ◽  
Mrna Levels

To investigate the presence and the distribution of preproendothelin-1 (prepro-ET-1) mRNA in human kidney, eight human kidneys obtained at surgery from patients affected by localized renal tumors were studied. Northern blot analysis using a human prepro-ET-1 cDNA probe labeled with 32P showed the presence of a single band of approximately 2.3 kb that was present both in the renal cortex and medulla of all the kidneys studied. Densitometric analysis of hybridization signals demonstrated that prepro-ET-1 mRNA levels in the renal medulla were 2.2-fold higher than those in the renal cortex. The distribution of prepro-ET-1 mRNA in human kidney was investigated by in situ hybridization using a human prepro-ET-1 RNA probe labeled with 35S. The greatest density of prepro-ET-1 mRNA was observed in the renal medulla, where hybridization signal was demonstrated in vasa recta bundles and capillaries and in collecting ducts. By combining in situ hybridization with immunohistochemical detection of von Willebrand factor, we demonstrated that 93 +/- 2.5% of nontubular medullary cells containing prepro-ET-1 mRNA were endothelial cells. In the cortex, prepro-ET-1 mRNA was localized in the endothelial layer of arcuate and interlobular arteries and veins and in the endothelial cells of afferent arterioles. The results of the present study demonstrate that ET-1 gene expression is present in vascular and tubular structures of the human kidney. It is possible that ET-1 synthesized locally in the human kidney represents a local system affecting renal hemodynamics and functions through paracrine and/or autocrine actions on different renal structures.

scholarly journals Tissue factor mRNA expression in the endothelium of an intact umbilical vein

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 174-179 ◽  
Author(s):  
MA Courtney ◽  
PJ Haidaris ◽  
VJ Marder ◽  
LA Sporn
Keyword(s):  
Endothelial Cells ◽  
In Situ Hybridization ◽  
Endothelial Cell ◽  
Mrna Expression ◽  
Tissue Factor ◽  
Umbilical Vein ◽  
Spotted Fever ◽  
Vascular Endothelial Cell ◽  
Immunocytochemical Staining

Abstract Tissue factor (TF) mRNA expression was measured by in situ hybridization in the endothelium of the intact human umbilical vein after infection with Rickettsia rickettsii. At 4 hours, R rickettsii organisms were clearly visible within approximately 70% of endothelial cells by immunocytochemical staining. Quantitation of TF mRNA expression revealed that the level within endothelial cells of the infected vein was significantly greater (3.7-fold, P < .0001) than that detected in uninfected endothelial cells. Serial sections of the umbilical cord vein were processed for in situ hybridization, and immunocytochemical staining and showed TF expression in those endothelial cells that contained R rickettsii organisms. Immunocytochemical staining for TF antigen at 6 hours was negative, but TF was clearly demonstrated within macrophages and fibroblasts of both control and infected umbilical cords. These studies demonstrate that the vascular endothelial cell, ex vivo, can be directly induced to express TF mRNA. This observation has not heretofore been clearly demonstrated except for in cultured endothelial cells. Since R rickettsii infection induces thrombotic vascular occlusions in patients with Rocky Mountain Spotted Fever, the results imply a potential role for endothelial cell TF in the pathogenesis of thrombotic disease.

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