[Warfarin can have a negative effect on bone formation].
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Adult↗
Biomedical subjects
K Blomgren
Publications and source records attributed to K Blomgren.
Calpastatin is up-regulated in response to hypoxia and is a suicide substrate to calpain after neonatal cerebral hypoxia-ischemia.
In a model of cerebral hypoxia-ischemia in the immature rat, widespread brain injury is produced in the ipsilateral hemisphere, whereas the contralateral hemisphere is left undamaged. Previously, we found that calpains were equally translocated to cellular membranes (a prerequisite for protease activation) in the ipsilateral and contralateral hemispheres. However, activation, as judged by degradation of fodrin, occurred only in the ipsilateral hemisphere. In this study we demonstrate that calpastatin, the specific, endogenous inhibitor protein to calpain, is up-regulated in response to hypoxia and may be responsible for the halted calpain activation in the contralateral hemisphere. Concomitantly, extensive degradation of calpastatin occurred in the ipsilateral hemisphere, as demonstrated by the appearance of a membrane-bound 50-kDa calpastatin breakdown product. The calpastatin breakdown product accumulated in the synaptosomal fraction, displaying a peak 24 h post-insult, but was not detectable in the cytosolic fraction. The degradation of calpastatin was blocked by administration of CX295, a calpain inhibitor, indicating that calpastatin acts as a suicide substrate to calpain during hypoxia-ischemia. In summary, calpastatin was up-regulated in areas that remain undamaged and degraded in areas where excessive activation of calpains and infarction occurs.
Animals↗
Spontaneous fracture of an ossified stylohyoid ligament.
The stylohyoid ligament extends from the styloid process to the hyoid bone. For an unknown reason it occasionally ossifies and forms a solid structure which can break because of trauma or even spontaneously. Symptoms of the fracture may mimic tumours, foreign bodies, infections or neuralgia. In our cases a spontaneous fracture of totally ossified stylohyoid ligaments presented as a painful neck swelling. The diagnosis was achieved by an ortopantomographic radiograph. In both cases the healing was spontaneous and complete.
Fractures, Spontaneous↗
Calpastatin is upregulated and acts as a suicide substrate to calpains in neonatal rat hypoxia-ischemia.
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Animals↗
Chemokine and inflammatory cell response to hypoxia-ischemia in immature rats.
Hypoxia-ischemia induces an inflammatory response in the immature central nervous system that may be important for development of brain injury. Recent data implicate that chemoattractant cytokines, chemokines, are involved in the recruitment of immune cells. The aim was to study alpha- and beta-chemokines in relation to the temporal activation of inflammatory cells after hypoxia-ischemia in immature rats. Hypoxia-ischemia was induced in 7-day-old rats (left carotid artery occlusion + 7.7% oxygen). The pups were decapitated at different times after the insult. Immunohistochemistry was used for evaluation of the inflammatory cell response and RT-PCR to analyze the cytokine mRNA and chemokine mRNA expression. A distinct interleukin-1beta and tumor necrosis factor-alpha cytokine expression was found 0-24 h after hypoxia-ischemia that was accompanied by induction of alpha-chemokines (growth related gene and macrophage inflammatory protein-2). In the next phase, the beta2-integrin expression was increased (12 h and onward) and neutrophils transiently invaded the vessels and tissue in the infarct region. The mRNA induction for the beta-chemokines macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, and RANTES preceded the expression of markers for lymphocytes [cluster of differentiation (CD)4, CD8], microglia/macrophages (MHC I), and natural killer cells in the infarct area. The activation of microglia/macrophages, CD4 lymphocytes, and astroglia persisted up to at least 42 d of postnatal age implicating a chronic component of immunoinflammatory activation. The expression of mRNA for alpha- and beta-chemokines preceded the appearance of immune cells suggesting that these molecules may have a role in the inflammatory response to insults in the immature central nervous system.
Animals↗
Antisense oligonucleotides to stromelysin mRNA inhibit injury-induced proliferation of arterial smooth muscle cells.
Smooth muscle cell migration and proliferation are important events in the formation of intimal lesions associated with atherosclerosis and restenosis following balloon angioplasty. To make this possible, the smooth muscle cell has to change from a contractile to an activated repair cell with capacity to synthesize DNA and extracellular matrix components. There is now considerable evidence that the extracellular matrix has important functions in modulating the phenotypic properties of smooth muscle cells, but less is known about the role of the matrix metalloproteinases. The present study investigates the role of stromelysin in the modulation of rat aortic smooth muscle cell morphology and function following mechanical injury in vitro and in vivo. Antisense mRNA oligonucleotides were used to investigate the role of stromelysin expression in injury-induced phenotypic modulation and the subsequent migration and proliferation of vascular smooth muscle cells. Cultured rat aortic smooth muscle cells and balloon-injured rat carotid arteries were used as experimental models. Light- and electron microscopy were used to follow changes in smooth muscle cell phenotype and lesion formation and incorporation of 3H-thymidine to detect DNA synthesis. Injury-induced DNA synthesis and migration in vitro were inhibited by 72% and 36%, respectively, by adding stromelysin antisense oligonucleotides to the medium prior to injury. In primary cultures, 67% of the smooth muscle cells treated with stromelysin antisense were retained in a contractile phenotype as judged by analysis of cell fine structure, compared to 15% untreated cells and 40% in cells treated with mismatched oligonucleotides. Examination of the carotid arteries one week after balloon injury likewise demonstrated a larger fraction of contractile cells in the inner parts of the media in vessels treated with antisense oligonucleotides compared to those treated with mismatched oligonucleotides. The neointima was also distinctly thinner in antisense-treated than in mismatched-treated and control arteries at this time. These findings indicate that stromelysin mRNA antisense oligonucleotides inhibited phenotypic modulation of rat arterial smooth muscle cells and so caused a decrease in migration and proliferation and neointima formation in response to vessel wall injury.
Animals↗
Effects of proteasome and calpain inhibitors on the structural reorganization and proliferation of vascular smooth muscle cells in primary culture.
Vascular smooth muscle cells exhibit a striking plasticity and are able to change from a differentiated, contractile phenotype to a more immature, synthetic phenotype. This includes a prominent structural reorganization with loss of myofilaments and construction of a large secretory apparatus. As a result, the cells lose their contractility and become able to migrate, proliferate, and secrete extracellular matrix components. In vivo, this phenotypic shift is a chief factor behind the involvement of smooth muscle cells in formation of atherosclerotic and restenotic lesions. Here, the effects of the proteasome inhibitors carbobenzoxy-leucyl-leucyl-leucinal, N-acetyl-leucyl-leucyl-norleucinal, and lactacystin on the morphologic structure and growth of rat aortic smooth muscle cells in primary culture were examined. Electron microscopic analysis revealed that the volume density of myofilaments was higher and the volume density of the endoplasmic reticulum and the Golgi complex was lower in cells exposed to these drugs than in solvent-treated controls. Moreover, diffuse material representing incompletely degraded proteins gathered in the cytoplasm of exposed cells. Similar material was also found in lysosomes. Immunogold staining showed a positive reaction in the diffuse cytoplasmic aggregates with antibodies against ubiquitin-protein conjugates and proteasomes, whereas the material collecting in lysosomes reacted only with those against ubiquitin-protein conjugates. Moreover, weak staining for smooth muscle alpha-actin was noted in the cytoplasmic aggregates. Otherwise, reactivity for this protein was concentrated in myofilaments. In addition to the effects on cell structure described above, the proteasome inhibitors blocked cell multiplication. This was probably due to a decreased rate of transition into a synthetic state as well as direct interference with cell cycle progression in synthetic cells. These observations suggest that proteasomes have the major responsibility for protein degradation during transition of smooth muscle cells from a contractile to a synthetic phenotype. If proteasome activity is inhibited, undegraded material accumulates in the cytoplasm and is only partially taken up into lysosomes for digestion. These findings raise the possibility that proteasome inhibitors may have a beneficial effect on vascular pathologies associated with phenotypic modulation and proliferation of smooth muscle cells.
Actins↗
The calpain proteolytic system in neonatal hypoxic-ischemia.
Neonatal rats were subjected to transient cerebral hypoxic-ischemia (HI, unilateral occlusion of the common carotid artery +7.70% O2 for 100 min) and allowed to recover for up to 14 days. Calpain caseinolytic activity was found to increase in both hemispheres for at least 20 hr. Hypoxic exposure per se increased the activity of calpains, more pronounced in a membrane-associated fraction, probably through interaction with cellular components, whereas HI introduced a loss of activity, most likely through consumption and loss of proteases. Consecutive tissue sections were stained with antibodies against calpastatin, alpha-fodrin, the 150-kDa breakdown product of alpha-fodrin (FBDP, marker of calpain proteolysis) or microtubule-associated protein 2 (MAP-2, marker of dendrosomatic neuronal injury). Areas with brain injury displayed a distinct loss of MAP-2, which clearly delineated the infarct. FBDP accumulated in injured and borderline regions ipsilaterally, and a less conspicuous, transient increase in FBDP also occurred in the contralateral hemisphere, especially in the white matter. The cytosolic fraction (CF) and the membrane and microsomal fraction (MMF) of cortical tissue were subjected to Western blotting and stained with antibodies against calpain, calpastatin and the 150-kDa breakdown product of alpha-fodrin (FBDP). Calpain immunoreactivity decreased bilaterally in the CF during the insult (62-68% of controls) and remained significantly lower during early recovery, whereas the MMF showed no significant changes. This translocation of calpains coincided with the appearance of FBDP in the ipsilateral, HI hemisphere, displaying a significantly higher level of FBDP from immediately after the insult until at least 1 day of recovery (204-292% of controls). No significant changes in FBDP were found in the contralateral, undamaged hemisphere, despite translocation of calpains in both hemispheres, a prerequisite for calpain activation. This discrepancy may be related to changes in the endogenous inhibitor, calpastatin. Calpastatin protein was found to decrease during and shortly after HI in the ipsilateral, but not the contralateral, hemisphere. The inhibitory activity of calpastatin also tended to decrease after HI, indicating that a reduction of calpastatin may be necessary for extensive calpain activation to occur. The mRNA of m-calpain increased in the HI hemisphere 48 hr after the insult (167%, p < 0.001), a time point when the protein was also increased. In summary, our findings indicate that calpains are activated during HI and in the early phase of reperfusion after HI, preceding neuronal death.
Animals↗
Expression of phenotype- and proliferation-related genes in rat aortic smooth muscle cells in primary culture.
OBJECTIVES: After endothelial injury, smooth muscle cells (SMCs) in the arterial media are modified from a contractile to a sympathetic phenotype. This process includes a prominent structural reorganization and makes the cells able to migrate into the intima, divide, and secrete extracellular matrix components. A similar change occurs in culture and then in vitro system has been established as a useful model in which to study the control of SMC differentiation. The purpose of this study was to analyze the expression of a number of phenotype- and proliferation-related genes in vascular SMCs during the first week in primary culture. METHODS: SMCs were enzymatically isolated from rat aorta and seeded on substrates of fibronectin (an adhesive plasma protein) and laminin-collagen type IV (two major basement membrane proteins) in a serum-free medium or in uncoated dishes in a serum-containing medium. Total RNA was isolated from the cells after different times of culture and analyzed by Northern blotting for expression of specific gene transcripts. In part, expression of the corresponding proteins was also explored by Western blotting and indirect immunofluorescence microscopy. RESULTS: The results indicate that the proto-oncogenes c-fos, c-jun and c-ets-1 were already activated during the isolation of the cells and then continued to be strongly expressed for a few days. Especially in the serum-free groups, there was also early activation of the genes for the matrix metalloproteinases, stromelysin (MMP-3) and type IV collagenase (MMP-2). In parallel, an increased expression of the genes for two extracellular matrix components was observed, with an early rise in osteopontin mRNA and a later rise in collagen type I mRNA. At the end of the test period, the corresponding proteins were deposited around the cells in a fibrillar pattern. Among the matrix receptors investigated, the beta 1 integrin subunit showed a high and persistent expression, whereas the alpha 5 and alpha 1 integrin subunits showed lower and more variable mRNA level. In support of the existence of an autocrine or paracrine platelet-derived growth factor (PDGF) loop, an early rise in expression of the PDGF A-chain gene and a subsequent rise in expression of the PDGF alpha-receptor gene were noted. CONCLUSION: It is proposed that the coordinated shift in gene expression here described to take place in connection with the phenotypic modulation of vascular SMCs in primary culture is part of a predetermined genetic program that normally serves the function to engage the cells in a wound healing response.
Animals↗
Phenotypic modulation of smooth muscle cells after arterial injury is associated with changes in the distribution of laminin and fibronectin.
Earlier in vitro studies suggest opposing roles of laminin and fibronectin in regulation of differentiated properties of vascular smooth muscle cells. To find out if this may also be the case in vivo, we used immunoelectron microscopy to study the distribution of these proteins during formation of intimal thickening after arterial injury. In parallel, cell structure and content of smooth muscle alpha-actin was analyzed. The results indicate that the cells in the normal media are in a contractile phenotype with abundant alpha-actin filaments and an incomplete basement membrane. Within 1 week after endothelial denudation, most cells in the innermost layer of the media convert into a synthetic phenotype, as judged by loss of actin filaments, construction of a large secretory apparatus, and destruction of the basement membrane. Some of these cells migrate through fenestrae in the internal elastic lamina and invade a fibronectin-rich network deposited on its luminal surface. Within another few weeks a thick neointima forms, newly produced matrix components replace the stands of fibronectin, and a basement membrane reappears. Simultaneously, the cells resume a contractile phenotype, recognized by disappearance of secretory organelles and restoration of alpha-actin filaments. These findings support the notion that laminin and other basement membrane components promote the expression of a differentiated smooth muscle phenotype, whereas fibronectin stimulates the cells to adopt a proliferative and secretory phenotype.
Actins↗
Expression of caveolae on the surface of rat arterial smooth muscle cells is dependent on the phenotypic state of the cells.
Both after vascular injury and when established in vitro, arterial smooth muscle cells pass through a characteristic change in phenotype. This process includes a prominent structural reorganization with partial loss of myofilaments and formation of a large endoplasmic reticulum and Golgi complex. As a result, the cells lose their contractility and become able instead to divide and to secrete extracellular matrix components. In the present study, the expression of plasma membrane caveolae in rat arterial smooth muscle cells was studied in primary culture and during the formation of neointimal thickenings after balloon injury. Electron microscopic analysis revealed that the number of caveolae (identified as flask-shaped invaginations of the plasma membrane) was reduced when the cells converted from a contractile to a synthetic phenotype (as defined morphologically) and then increased again when they readopted a more differentiated state. However, immunoblotting analysis did not show any changes in the cellular content of caveolin (a major protein component of caveolae) during the 1st week in culture. At the same time, immunocytochemical staining demonstrated a shift in the localization of caveolin from small spot-like structures dispersed over the cell surface to vesicular structures in the perinuclear cytoplasm. These findings indicate that the transition of smooth muscle cells from a contractile to a synthetic phenotype involves a marked decline in the number of plasma membrane caveolae. In parallel, caveolin is internalized and redistributed to Golgi-associated vesicles in the perinuclear cytoplasm. In context of the rapidly increasing awareness of the importance of caveolae both in signal transduction and intracellular cholesterol transport, it seems likely that the variations in the number of caveolae may be significant for the functional differences between smooth muscle cells in different phenotypes.
Animals↗
[New guidelines from the Medical Products Agency: the triple regime against Helicobacter pylori is necessary. Treat only if the ulcer is verified].
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Amoxicillin↗
Degradation of fodrin and MAP 2 after neonatal cerebral hypoxic-ischemia.
Neonatal rats were subjected to transient cerebral hypoxic-ischemia (unilateral occlusion of the common carotid artery + 7.70% O2 for 100 min) and allowed to recover for 3 h, 24 h, 2 days or 14 days. Consecutive tissue sections were stained with antibodies against alpha-fodrin, the 150 kDa breakdown product of alpha-fodrin (FBDP, marker of calpain proteolysis) or microtubule associated protein 2 (MAP 2, marker of dendrosomatic neuronal injury). Cortical tissue pieces were subjected to Western blotting using the antibody against the FBDP. Areas with brain injury displayed a distinct loss of MAP 2 which clearly delineated the infarct. FBDP accumulated in injured and borderline regions ipsilaterally and a less conspicuous, transient increase in FBDP also occurred in the contralateral hemisphere, especially in the white matter. A reciprocal staining pattern could be seen in the cerebral cortex, i.e. loss of MAP 2 and accumulation of FBDP, most pronounced 14 days after the insult. Fodrin and MAP 2 are known calpain substrates, and degradation of these proteins preceded neuronal degeneration, indicating that these proteases may be involved in the early events triggering the cascades leading to neuronal death.
Animals↗
Fodrin degradation and subcellular distribution of calpains after neonatal rat cerebral hypoxic-ischemia.
Neonatal rats were subjected to transient cerebral hypoxic-ischemia (unilateral occlusion of the common carotid artery + 7.70% O2 for 100 min). Ipsi-and contralateral parietal cerebral cortex was assayed with Western blotting for fodrin breakdown product (FBDP). Calpain immunoreactivity was assayed in the cytosolic fraction (CF) and the membrane and microsomal fraction (MMF). Calpain immunoreactivity decreased bilaterally in the CF during the insult (62-68% of controls) and remained significantly lower during early recovery, whereas the MMF showed no significant changes. This relative redistribution of calpains coincided with the appearance of FBDP in the left, ipsilateral hemisphere, displaying a significantly higher level of FBDP from immediately after the insult until at least 1 day of recovery (204-292% of controls). No significant changes in FBDP could be detected in the right, contralateral hemisphere, indicating that although redistribution of calpains occurred, hypoxia per se did not suffice to initiate fodrin degradation in this model of neonatal hypoxic-ischemia.
Animals↗
Phenotypic modulation of smooth muscle cells during the formation of neointimal thickenings in the rat carotid artery after balloon injury: an electron-microscopic and stereological study.
The formation of neointimal thickenings in the rat carotid artery after balloon injury was studied by a combination of electron-microscopic and stereological methods. All smooth muscle cells in the normal media had a contractile phenotype, the cytoplasm being dominated by myofilaments. Seven days after endothelial denudation, the smooth muscle cells in the innermost part of the media had assumed a synthetic phenotype by loss of myofilaments and formation of a large endoplasmic reticulum and Golgi complex. These cells moved through fine openings in the internal elastic lamina and gave rise to a growing neointima by proliferation and secretion of extracellular matrix components. Fourteen days after the operation, the neointima had almost reached its final size, and mitoses were no longer noted. Nevertheless, the cells maintained a synthetic phenotype with prominent secretory organelles, although myofilaments had started to become more abundant again. They were surrounded by an extracellular matrix made up of collagen fibrils and coalescing patches of elastin. Thirty-five days after the operation, an endothelial cell layer had reformed and covered most of the luminal vessel surface. In parallel, the smooth muscle cells in the neointima had returned to a contractile phenotype with a cytoplasm dominated by myofilaments. These findings provide a morphological basis for further analysis of the cellular and molecular interactions involved in the formation of neointimal thickenings after endothelial injury, and for the search for agents interfering with this process.
Animals↗
Calpain and calpastatin activity in the optic pathway.
The levels of the neutral proteolytic enzymes calpains and their endogenous inhibitor calpastatin were determined in the retina and in the retrobulbar optic pathway in the albino rabbit. The highest level of calpains was observed in the optic nerve with decreasing levels in the optic tract and superior colliculus. The level of calpastatin in the retina was very low compared to that in the optic nerve and tract and other parts of the nervous system.
Animals↗
Phenotype modulation in primary cultures of rat aortic smooth muscle cells. Effects of drugs that interfere with the functions of the vacuolar system and the cytoskeleton.
The transition of adult rat aortic smooth muscle cells from a contractile to a synthetic phenotype during the first week of primary culture on a substrate of fibronectin in serum-free medium was studied by light and electron microscopy. The weak base chloroquine and the carboxylic ionophore monensin were both found to inhibit the spreading of the cells and the accompanying changes in cellular fine structure. The exchange of myofilament bundles for a prominent rough endoplasmic reticulum and Golgi complex was delayed and vacuoles filled with incompetely degraded material accumulated in the cytoplasm. The microtubule-disruptive drugs colchicine and nocodazole likewise opposed the spreading and fine structural reorganization of the cells. Most typically, the Golgi stacks were small and widely dispersed. In addition, vacuoles of the type mentioned above increased in number. On the other hand, there was surprisingly little effect of cytochalasin B, a drug that is supposed to interfere with the assembly of actin filaments. The observations suggest that the phenotypic modulation of arterial smooth muscle cells is dependent on: (a) lysosomal degradation of discarded cellular constituents, (b) active vesicular transport along the exocytic pathway to provide the expanding cell surface with new membrane, and (c) a normal microtubular cytoskeleton to ensure the establishment of a new and functionally efficient intracellular organization.
Animals↗
Developmental changes of calpain and calpastatin in rabbit brain.
A major part of the Ca-activated proteolytic activity in the soluble fraction from rabbit brain could be due to the activity of the neutral thiol-proteases calpain I and II. The activity of calpains exceeded that of the endogenous inhibitor, calpastatin, at all developmental stages studied. The level of calpains increased rapidly from the prenatal stage to reach a peak 10-20 days postnatally. From this period the level of calpains decreased slowly to reach the adult levels. The level of calpastatin increased steadily from the prenatal stage to old age.
Aging↗