Increased medial smooth muscle cell length is responsible for vascular hypertrophy in young hypertensive rats

Large mesenteric arteries from 3- to 4-wk-old spontaneously hypertensive rats (SHR) showed medial hypertrophy and an increased contractile response to various agonists before significant blood pressure increase. Here we determined the cellular nature of this vascular hypertrophy. Large mesenteric arteries from SHR and Wistar-Kyoto (WKY) rats were fixed at maximal relaxation either with an in situ perfusion fixation or an in vitro fixation method. With the use of morphometric protocols and confocal microscopy, the volume of the medial wall and lumen, numerical density of smooth muscle cell nuclei in the medial layer, and smooth muscle cell and nuclear length were measured. Both methods of fixation yielded similar results, showing significant medial volume expansion in SHR than WKY without lumen change. Numerical density of medial smooth muscle cells was significantly less in SHR than WKY, and their total number per 100 microm length were similar between the strains. Average smooth muscle nuclear and cell length from SHR was significantly longer than that of WKY. Regression analysis showed that the increase in smooth muscle cell length explained 80% of the medial volume increase. We concluded that increased smooth muscle cell length in prehypertensive SHR is responsible for increased medial volume in the mesenteric arteries.     

Mimecan is involved in aortic hypertrophy induced by sinoaortic denervation in rats

This study was designed to investigate whether mimecan was involved in aortic hypertrophy induced by sinoaortic denervation in rats. 8 weeks after sinoaortic denervation, when compared to sham-operated rats, sinoaortic denervated rats exhibited aortic hypertrophy and down-regulation of mimecan. Through classic univariate correlation analysis, it was found that mimecan mRNA was negatively related to extent of aortic hypertrophy. Treatment of primary cultured vascular smooth muscle cells with the Ang II (1 μM), which was found locally increased in the aortae of sinoaortic denervated rats, resulted in a reduction of mimecan expression. In vitro, knockdown of mimecan in vascular smooth muscle cells promoted cell proliferation induced by 15% of fetal bovine serum or Ang II (1 μM). We concluded that down-regulation of mimecan was involved in aortic hypertrophy induced by sinoaortic denervation in rats.     

Neuroplasticity in the smooth muscle of the myenterically and extrinsically denervated rat jejunum

The objective of this study was to examine the effects of two different denervation procedures on the distribution of nerve fibers and neurotransmitter levels in the rat jejunum. Extrinsic nerves were eliminated by crushing the mesenteric pedicle to a segment of jejunum. The myenteric plexus and extrinsic nerves were eliminated by serosal application of the cationic surfactant benzyldimethyltetradecylammonium chloride (BAC). The effects of these two denervation procedures were evaluated at 15 and 45 days. The level of norepinephrine in whole segments of jejunum was initially reduced by more than 76% after both denervation procedures, but by 45 days the level of norepinephrine was the same as in control tissue. Tyrosine hydroxylase (nor-adrenergic nerve marker) immunostaining was absent at 15 days, but returned by 45 days. However, the pattern of noradrenergic innervating axons was altered in the segment deprived of myenteric neurons. Immunohistochemical studies showed protein gene product 9.5 (PGP 9.5)-immunoreactive fibers in whole-mount preparations of the circular smooth muscle in the absence of the myenteric plexus and extrinsic nerves. At 45 days, the number of nerve fibers in the circular smooth muscle increased. Vasoactive intestinal polypeptide (VIP)-immunoreactive fibers, a subset of the PGP 9.5 nerve fibers, were present in the circular smooth muscle at both time points examined. Choline acetyltransferase (CAT) activity and VIP and leucine enkephalin levels were measured in separated smooth muscle and submucosa-musosal layers of the denervated jejunum. VIP and leucine-enkephalin levels were no different from control in tissue that was extrinsically denervated alone. However, the levels of these peptides were elevated two-fold in the smooth muscle 15 and 45 days after myenteric and extrinsic denervation. In the submucosa-mucosa, VIP and leucine enkephalin levels also were elevated two-fold at 15 days, but comparable to control at 45 days. CAT activity was equal to control in the smooth muscle but elevated two-fold in the submucosa-mucosa at both times. These results provide evidence for innervation of the circular smooth muscle by the submucosal plexus. Moreover, these nerve fibers originating from the submucosal plexus proliferate in the absence of the myenteric plexus. Furthermore, the myenteric neurons appear to be essential for normal innervation of the smooth muscle by the sympathetic nerve fibers. It is speculated that the sprouting of the submucosal plexus induced by myenteric plexus ablation is mediated by increased production of trophic factors in the hyperplastic smooth muscle.     

Morphometry of the amount of smooth muscle cells in the media of various rabbit arteries

Our objective in this study was to evaluate the relative amount of smooth muscle cells in the medial layer of various rabbit arteries. The fixation of smooth muscle cells in the arterial wall is difficult and the differential effect of glutaraldehyde (GA) and fixative vehicle on cell ultrastructure in different tissues is controversial. We compared the effect of various concentrations of the vehicle and glutaraldehyde (osmolarity ranges for total fixative, 350-1030 mOsm) on the arterial wall ultrastructure. We found that a 600 mOsm GA solution (isotonic vehicle; 2.5% GA) adequately preserves arterial wall structures. The relative amount of smooth muscle cells in the media differed in various segments along the arterial tree. It ranged from 35% (thoracic aorta) to 74% (tibial artery). The importance of weighting the contractile response of different arteries in vitro to their relative smooth muscle cell content is discussed.     

Diversity of vinculin/meta-vinculin in human tissues and cultivated cells. Expression of muscle specific variants of vinculin in human aorta smooth muscle cells

Microheterogeneity of different vinculin and meta-vinculin isoforms in adult human tissues and cultured cells was studied by two-dimensional gel electrophoresis and immunoblotting technique. Four isoforms of vinculin (alpha, alpha', beta, and gamma) and two isoforms of meta-vinculin (alpha and beta) were resolved. alpha-, alpha'-, and beta-isoforms of vinculin were found in all cell types and tissue samples analyzed in the present study. gamma-Isoform of vinculin and both alpha- and beta-isoforms of meta-vinculin were found in smooth (aorta wall and myometrium) and cardiac muscle, rather than in skeletal muscle, liver, foreskin fibroblasts, and macrophages. In the primary culture of human aorta smooth muscle cells, the fractional content of gamma-isoform of vinculin and meta-vinculin was dramatically reduced, and, by the onset of intensive cell division, the proteins could hardly be detected. Subcultured human aorta smooth muscle cells did not contain gamma-vinculin and meta-vinculin. We analyzed the microheterogeneity of vinculin and meta-vinculin in three smooth muscle layers of human aorta wall--media, muscular-elastic (adjacent to media) intima, and subendothelial (juxtaluminal) intima. It was shown that in media the fractional content of gamma-isoform of vinculin was 45% and meta-vinculin, 42%; in muscular-elastic intima the fractional content of gamma-vinculin was 42% and meta-vinculin, 36%. However, in subendothelial intima, the share of these proteins was significantly lower than in adjacent muscular-elastic intima and media. Isoactin pattern that is characteristic of smooth muscle was identical in all aortic layers, thus proving the smooth muscle origin of subendothelial intima cells. These findings demonstrate that human aortic smooth muscle cells in vivo and in vitro undergo coordinated differential expression of smooth muscle specific variants of vinculin, i.e. gamma-vinculin and meta-vinculin.     

Neural Control of Muscle Androgen Receptors

The number of cytosolic androgen receptors in rat skeletal muscle increases following denervation and disuse. This increase was postulated to represent altered intracellular distribution and consequent diminished sensitivity of skeletal muscle to androgens. To test this hypothesis, we measured total (homogenate) androgen receptor levels after denervation. Total (homogenate) androgen receptor binding did not change in response to denervation of leg muscles from adult male rats. An increase in cytosolic receptor number with no increase in total (homogenate) receptor levels supports the hypothesis of altered intracellular distribution of androgen receptors in denervated muscle. Cytosolic androgen receptor binding in muscle from male rats increased by 40% after denervation, whereas in females the increase was 17%. These increases could not be altered by endocrine manipulations of males or females.     

Changes of opening angle in hypertensive and hypotensive arteries in 3-day organ culture

To study the effect of pressure changes on the opening angle of arteries in organ culture, tubular segments of porcine common carotid arteries were cultured with pulsatile flow perfusion under hypertensive (150+/-20 mmHg), normotensive (100+/-20 mmHg), or hypotensive (30+/-10 mmHg) pressure while maintaining the arteris at a physiological wall shear stress of approximately 15 dyn/cm(2) for up to 3 days. Arteries were then cut into short ring segments by sections perpendicular to the axis and then cut open radially to observe the opening angle in aerated phosphate buffered saline solution (37 degrees C). Norepinephrine (NE, 10 microM), carbacol (CCh, 100 microM), and sodium nitroprusside (SNP, 10 microM) were added after the radial cut at 30, 20, and 30 min intervals, the opening angles were measured, respectively. Results show that hypertensive arteries developed a significantly larger opening angle than normotensive and hypotensive arteries, associated with a significant increase in cell proliferation. In addition, with smooth muscle contraction activated by NE, the opening angle decreases significantly in hypertensive arteries but has little change in hypotensive and normotensive arteries, indicating an enhancement of smooth muscle contraction on the lumen side of the hypertensive arterial wall. In comparison, hypotensive pressure has little effect on arterial opening angle and cell proliferation.     

Difference in the ability of neonatal and adult denervated muscle to accumulate acetylcholinesterase at the old sites of innervation

In adult rat sternocleidomastoid muscle, AChE is concentrated in the region rich in motor end-plates (MEP). All major AChE forms, "16 S," "10 S," and "4 S," are accumulated at high levels, and not only "16 S" AChE. After denervation, muscle AChE decreases; 2 weeks after denervation, low levels (20-40% of control) are reached for all forms. During the following weeks, a slow but steady increase in "10 S" and "16 S" AChE occurs in the denervated muscle. At this stage, all forms are again observed to be highly concentrated in the region containing the old sites of innervation. Thus, in adult rat muscle the structures able to accumulate "16 S," "10 S," and "4 S" AChE in the MEP-rich regions remain several months after denervation. In normal young rat sternocleidomastoid muscle at birth, all AChE forms are already accumulated in the MEP-rich region. After denervation at birth, the denervated muscle loses its ability to keep a high concentration of "4 S," "10 S," and "16 S" AChE in the old MEP-rich region. All AChE forms are still present 1 month after denervation, but they are decreased and diffusedly distributed over the whole length of the muscle. In particular, "16 S" AChE is detected in the same proportion (10-15%) all along the denervated muscle. Thus, the diffuse distribution of AChE, and especially "16 S" AChE, after neonatal denervation, contrasts with the maintained accumulation observed in adult denervated muscle. It seems that denervation of young muscle results in a specific loss of the muscle ability to concentrate high levels of all AChE forms at the old sites of innervation.     

Influence of chronic nadolol treatment on blood pressure and vascular changes in spontaneously hypertensive rats.

Chronic treatment of spontaneously hypertensive rats (SHR) and Kyoto-Wistar normotensive rats (WKY) with nadolol was carried out from gestation until 28 weeks of age. Nadolol treatment caused some lowering of blood pressure but did not prevent the development of hypertension or cardiac hypertrophy in the SHR, in spite of significant beta-blockade. The lumen of large mesenteric arteries from control SHR was smaller than from WKY, and nadolol treatment increased the lumen size in the SHR. An increased number of smooth muscle cell layers present in the control SHR as compared with WKY was reduced slightly by nadolol treatment. However, the changes produced by nadolol did not reach the levels of control and treated WKY. In the aorta, the incidence of polyploid smooth muscle cells was higher in the SHR than the WKY in the control group. Nadolol treatment reduced the percentage of polyploid cells in both SHR and WKY, so that the difference between these two groups of animals was eliminated in the treated groups. The tissue level of norepinephrine in the plasma, heart, mesenteric arteries, and adrenal glands in the SHR and WKY was not affected by the treatment. We suggest that the ineffectiveness of nadolol in preventing hypertension development may be due to its lack of effect in preventing primary changes in the resistance arteries, and that the development of polyploidy of smooth muscle cells may be mediated by beta-receptors.     

The effects of denervation on protein turnover of the soleus and extensor digitorum longus muscles of adult mice.

1. Changes in protein turnover of the soleus and EDL muscles of adult mice have been studied 1, 7 and 80 days after denervation. 2. Increased rates of protein degradation 7 and 80 days post-denervation correlated with the atrophy and loss of protein from these muscles. 3. Rates of protein synthesis in the EDL decreased 24 hr after nerve section. However, these synthetic rates increased again to become higher in the 7 day denervated muscles compared with their controls. These latter anabolic changes are inconsistent with the concept of a denervated muscle being inactive. 4. These findings have been compared with a similar study on muscles of growing rats. Any passive stretching of the denervated muscles by continued bone growth appears unlikely to be a crucial factor explaining the increased rates of protein synthesis 7 days after denervation.     

Ovine middle cerebral artery characterization and quantification of ultrastructure and other features: changes with development

Regulation of tone, blood pressure, and blood flow in the cerebral vasculature is of vital importance, particularly in the developing infant. We tested the hypothesis that, in addition to accretion of smooth muscle cells (SMCs) in cell layers with vessel thickening, significant changes in smooth muscle structure, as well as phenotype, extracellular matrix, and membrane proteins, in the media of cerebral arteries (CAs) during the course of late fetal development account for associated changes in contractility. Using transmission electron, confocal, wide-field epifluorescence, and light microscopy, we examined the structure and ultrastructure of CAs. Also, we utilized wire myography, Western immunoblotting, and real-time quantitative PCR to examine several other features of these arteries. We compared the main branch ovine middle CAs of 95- and 140-gestational day (GD) fetuses with those of adults (n = 5 for each experimental group). We observed a graded increase in phenylephrine- and KCl-induced contractile responses with development. Structurally, lumen diameter, media thickness, and media cross-sectional area increased dramatically from one age group to the next. With maturation, the cross-sectional profiles of CA SMCs changed from flattened bands in the 95-GD fetus to irregular ovoid-shaped fascicles in the 140-GD fetus and adult. We also observed a change in the type of collagen, specific integrin molecules, and several other parameters of SMC morphology with maturation. Ovine CAs at 95 GD appeared morphologically immature and poorly equipped to respond to major hemodynamic adjustments with maturation.     

Satellite cells on isolated myofibers from normal and denervated adult rat muscle.

Satellite cells (SCs) in normal adult muscle are quiescent. They can enter the mitotic program when stimulated with growth factors such as basic FGF. Short-term denervation stimulates SC to enter the mitotic cycle in vivo, whereas long-term denervation depletes the SC pool. The molecular basis for the neural influence on SCs has not been established. We studied the phenotype and the proliferative capacity of SCs from muscle that had been denervated before being cultured in vitro. The expression of PCNA, myogenin, and muscle (M)-cadherin in SCs of normal and denervated muscle fibers was examined at the single-cell level by immunolabeling in a culture system of isolated rat muscle fibers with attached SCs. Immediately after plating (Day 0), neither PCNA nor myogenin was present on normal muscle fibers, but we detected an average of 0.5 M-cadherin(+) SCs per muscle fiber. The number of these M-cadherin(+) cells (which are negative for PCNA and myogenin) increased over the time course examined. A larger fraction of cells negative for M-cadherin underwent mitosis and expressed PCNA, followed by myogenin. The kinetics of SCs from muscle fibers denervated for 4 days before culturing were similar to those of normal controls. Denervation from 1 to 32 weeks before plating, however, suppressed PCNA and myogenin expression almost completely. The fraction of M-cadherin(+) (PCNA(-)/myogenin(-)) SCs was decreased after 1 week of denervation, increased above normal after denervation for 4 or 8 weeks, and decreased again after denervation for 16 or 32 weeks. We suggest that the M-cadherin(+) cells are nondividing SCs because they co-express neither PCNA or myogenin, whereas the cells positive for PCNA or myogenin (and negative for M-cadherin) have entered the mitotic cycle. SCs from denervated muscle were different from normal controls when denervated for 1 week or longer. The effect of denervation on the phenotypic modulation of SCs includes resistance to recruitment into the mitotic cycle under the conditions studied here and a robust extension of the nonproliferative compartment. These characteristics of SCs deprived of neural influence may account for the failure of denervated muscle to fully regenerate. (J Histochem Cytochem 47:1375-1383, 1999)     

Hypoxic remodeling of the rat pulmonary arterial microcirculation assessed by microdissection.

Artery segments were microdissected from distal acini of the rat lung and studied by light and electron microscopy. Morphometric methods were used to quantify the structure of the wall at defined levels within the normal axial pathway and to determine the changes after 5 and 7 days of whole-animal exposure to hypobaric hypoxia at an inspiratory O2 fraction of 0.1. In the normal lung, at the level of the terminal bronchiolus, the artery wall comprised up to six layers of smooth muscle cells (SMCs). At the respiratory bronchiolar level, however, the wall contained fewer than two layers of SMCs with a consistently circumferential orientation. By the second-generation alveolar ducts (AD2), the medial layer was lost, replaced by subendothelial precursor smooth muscle cells (PCs) resembling intermediate cells. At this and the next level (AD3), the PC layer was often circumferentially discontinuous. Regression analysis of the morphometric data suggested, however, that the smallest AD3 artery is likely to have a layer of PCs but with virtually no measurable separation between them and the endothelium. The mean maximum radial diameter of SMCs decreased along the axial pathway with a significant difference between diameters at terminal bronchiolus and AD2 levels; yet the diameter of endothelial cells remained the same. After 7 days of hypoxia, no change was noted in the number of smooth muscle layers, but at the AD2 level the relative area of media in the total wall increased. This was due in part to hypertrophy of PCs, as evidenced by an increase in their mean maximum radial diameter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neural influence on the expression of acetylcholinesterase molecular forms in fast and slow rabbit skeletal muscles

With the aim of investigating the roles of motor innervation and activity on muscle characteristics, we studied the molecular forms of acetylcholinesterase (AChE) in fast-twitch (semimembranosus accessorius; SMa) and slow-twitch (semimembranosus proprius; SMp) muscles of the rabbit. We have shown that SMa and SMp express different patterns and tissue distribution of AChE forms and that the effect of long denervation varies with age. Three principal findings concerning expression of AChE molecular forms emerge from these studies. (1) The activity of AChE and the pattern of its molecular forms are particularly altered in adult denervated SMa and SMp muscles. AChE activity increases by 10-fold in both muscles, but asymmetric forms disappear in SMa and increase by 20-fold in SMp muscles. A similar alteration of AChE is found after tenotomy of these muscles, showing that the effect of denervation may be partly due to suppression of muscle activity. (2) The different changes occurring in the composition of AChE molecular forms in adult denervated SMa and SMp muscles are consistent with fluorescent staining with anti-AChE monoclonal antibodies and with DBA or VVA lectins, which bind to AChE asymmetric, collagen-tailed forms. These lectins poorly stain denervated SMa muscle surfaces but intensely stain neuromuscular junctions and extrasynaptic areas in denervated SMp muscle. (3) In contrast with the adult, denervation of 1-day-old muscles does not markedly modify the total amount of AChE or the proportions of its molecular forms, despite dramatic effects on muscle structure. These results are supported by studies of labeling with fluorescent DBA: the lectin only slightly stains the muscle fiber surface of denervated 15-day-old SMp muscle. Taken together, these data show that denervated muscles escape physiological regulation, producing increased levels of AChE with highly variable cellular distribution and patterns of molecular forms, depending on the age of operation and on the type of muscle.     

Proteomic analysis of rat laryngeal muscle following denervation

Laryngeal muscle atrophy induced by nerve injury is a major factor contributing to the disabling symptoms associated with laryngeal paralysis. Alterations of global proteins in rat laryngeal muscle following denervation were, therefore, studied using proteomic techniques. Twenty-eight adult Sprague-Dawley rats were divided into normal control and denervated groups. The thyroarytenoid (TA) muscle was excised 60 days after right recurrent laryngeal nerve was resected. Protein separation and identification were preformed using 2-DE and MALDI-MS with database search. Forty-four proteins were found to have significant alteration in expression level after denervation. The majority of these proteins (57%), most of them associated with energy metabolism, cellular proliferation and differentiation, signal transduction and stress reaction, were decreased levels of expression in denervated TA muscle. The remaining 43% of the proteins, most of them involved with protein degradation, immunoreactivity, injury repair, contraction, and microtubular formation, were found to have increased levels of expression. The protein modification sites by phosphorylation were detected in 22% of the identified proteins that presented multiple-spot patterns on 2-D gel. Significant changes in protein expression in denervated laryngeal muscle may provide potential therapeutic strategies for the treatment of laryngeal paralysis.     

Proliferation of arteriovenous anastomoses in the developing rabbit ear is enhanced after denervation

The innervation of the rabbit ear vasculature by noradrenergic and substance P-immunoreactive (SP-IR) nerves was investigated in both young and adult animals. All vascular segments were supplied by both noradrenergic and SP-IR nerves. In the ear margins, the arteriovenous anastomoses (AVAs) were more densely innervated than other vessels. In general, the density of both noradrenergic and SP-IR nerves increased with age until 10-12 weeks postnatum. The ear vasculature was denervated in young rabbits to test whether the normal proliferation of AVAs in the growing ear was altered in the absence of nerves. Surgical resection of all auricular nerve trunks except the auricular branch of the auriculo-temporal nerve, and removal of the ipsilateral superior cervical ganglion in 10- to 12-day-old rabbits left the ear devoid of both noradrenergic and SP-IR nerves for at least four weeks. Repeated administration of 6-hydroxydopamine (6-OHDA) to young rabbits produced degeneration of noradrenergic terminal axons, but preterminal nerve trunks survived. SP-IR nerves did not appear to be affected by 6-OHDA. These denervation regimes resulted in a 1.5- to 2-fold increase in the number of AVAs formed in the growing ear. Surgical denervation and 6-OHDA treatment also led to retarded growth of the media of the central ear artery, but this effect of 6-OHDA was probably not due to a specific action on vascular smooth muscle. Surgical resection of most of the dorsal auricular nerves in adult rabbits did not affect AVA density. Large differences in AVA density were apparent between groups of control animals from different parts of the country, or groups examined at different times of the year. These results demonstrate that the labile nature of AVAs in the rabbit ear can result in considerable variability in the absolute number of AVAs and suggest that both intrinsic and extrinsic factors may influence development of the microvasculature.