Phenotypic modulation of smooth muscle cells in lymphoedema.

BACKGROUND: Lymphoedema is a debilitating progressive condition that is frequently observed following cancer surgery and severely restricts quality of life. Although it is known that lymphatic dysfunction and obstruction underlie lymphoedema, the pathogenic mechanism is poorly understood. Smooth muscle cells (SMCs) play pivotal roles in the pathogenesis of various vascular diseases, including atherosclerosis. OBJECTIVES: We analysed SMCs in lymphatic vessels from the lymphoedematous legs of 29 patients. METHODS: Expression of smooth muscle α-actin (SMαA) and smooth muscle myosin heavy chain (SM-MHC) isoforms SM1 and SM2 was investigated using immunohistochemistry. RESULTS: Compared with normal lymphatic vessels, all affected lymphatic vessels in chronic lymphoedema showed marked wall thickening. In addition to increases in the numbers of rows of SMαA(+) SM1(+) SMCs in the tunica media, SMCs were also observed in the subendothelial region (tunica intima). While most intimal and medial cells were positive for SMαA and SM1, staining for SM1 and particularly SM2, a marker of mature SMCs, progressively declined in lymphatic vessels in increasingly severe lymphoedema lesions. Consequently, the SM1(+) and SM2(+) cell fractions were significantly reduced in the tunica media and intima of lymphatic vessels. CONCLUSIONS: These observations indicate that the lymphatic tunica media and tunica intima consist mainly of phenotypically modulated SMCs, and that SMCs play a key role in the development of lymphoedema.

Macrophages and islet inflammation in type 2 diabetes.

Type 2 diabetes (T2D) is characterized by insulin resistance and impaired insulin secretion from pancreatic ß-cells. Inflammatory cytokines, including tumour necrosis factor-α (TNF-α), have been shown to promote insulin resistance, and altered expression of cytokines (adipokines) in obese adipose tissue is thought to be an important link between obesity and insulin resistance. It is also becoming clear that inflammation plays a key role in the development of ß-cell dysfunction. Inflammatory changes, including accumulation of macrophages, have been documented in T2D islets. Moreover, therapeutic inhibition of interleukin-1ß (IL-1ß) ameliorates ß-cell dysfunction in humans. This review summarizes current understanding of the molecular mechanisms underlying inflammation within islets and its relation to ß-cell dysfunction in T2D. A particular focus is on the physiological and pathological functions of macrophages within islets.

CArG elements control smooth muscle subtype-specific expression of smooth muscle myosin in vivo.

Expression of smooth muscle myosin heavy chain (SM-MHC) is tightly controlled depending on the differentiated state of smooth muscle cells (SMCs). To better understand the mechanisms that regulate transcription of the SM-MHC gene in vivo, we tested the function of several conserved CArG elements contained within the -4200 to +11,600 region of this gene that we had previously shown to drive SMC-specific expression in transgenic mice. CArG1 in the 5'-flanking sequence was required for all SMCs, while CArG2 and a novel intronic CArG element were differentially required in SMC subtypes. Of particular note, mutation of the intronic CArG selectively abolished expression in large arteries. A promoter construct containing three repeats of a conserved 227-bp intronic CArG-containing region was sufficient to direct transcription in vascular SMCs in transgenic mice, although this construct was also expressed in skeletal and cardiac muscle. These results support a model in which transcriptional regulation of SM-MHC is controlled by multiple positive and negative modular control regions that differ between SMCs and non-SMCs and among SMC subtypes. We also demonstrated that the CArG elements of the endogenous SM-MHC gene were bound by SRF in chromatin.

Recruitment of serum response factor and hyperacetylation of histones at smooth muscle-specific regulatory regions during differentiation of a novel P19-derived in vitro smooth muscle differentiation system.

Little is known regarding transcriptional regulatory mechanisms that control the sequential and coordinate expression of genes during smooth muscle cell (SMC) differentiation. To facilitate mechanistic studies of SMC differentiation, we established a novel P19-derived clonal cell line (designated A404) harboring a smooth muscle (SM) alpha-actin promoter/intron-driven puromycin resistance gene. Retinoic acid plus puromycin treatment stimulated rapid differentiation of multipotential A404 cells into SMCs that expressed multiple SMC differentiation marker genes, including the definitive SM-lineage marker SM myosin heavy chain. Using this system, we demonstrated that various transcription factors were upregulated coincidentally with the expression of SMC differentiation marker genes. Of interest, the expression of serum response factor (SRF), whose function is critical for SMC-specific transcription, was high in undifferentiated A404 cells, and it did not increase over the course of differentiation. However, chromatin immunoprecipitation analyses showed that SRF did not bind the target sites of endogenous SMC marker genes in chromatin in undifferentiated cells, but it did in differentiated A404 cells, and it was associated with hyperacetylation of histones H3 and H4. The present studies define a novel cell system for studies of transcriptional regulation during the early stages of SMC differentiation, and using this system, we obtained evidence for the involvement of chromatin remodeling and selective recruitment of SRF to CArG elements in the induction of cell-selective marker genes during SMC differentiation.

Development of a smooth muscle-targeted cre recombinase mouse reveals novel insights regarding smooth muscle myosin heavy chain promoter regulation.

The use of genetically modified mice has been an important model system to study gene function in cardiovascular development and under pathophysiological conditions. Although conventional gene knockout studies have provided important insights into gene function in the cardiovascular system, they may be limited by upregulation of compensatory pathways and the inability to differentiate direct versus indirect functions in vivo. As a first step in developing systems that can target gene activation or inactivation specifically to smooth muscle cells (SMCs), we coupled the smooth muscle myosin heavy chain (SMMHC) promoter to the cre recombinase gene and generated transgenic mice that express cre in SMCs. In addition, we used these mice to address whether the heterogeneous staining observed in SMMHC-LacZ mice was due to subsets of SMCs that required different regulatory cassettes of the promoter or if it reflected episodic expression of the transgene. To address both the feasibility of SMC targeting and the apparent heterogeneous expression, we bred SMMHC-cre mice to indicator mice containing a cre-activated LacZ gene. Results showed high-level expression in SMCs at various embryonic time points and in adult tissues. Because breeding of SMMHC-cre mice to an indicator line provided an integration of cre activity over time, results of this study revealed that expression of the SMMHC promoter fragment more closely resembled the expression of the endogenous gene, both with respect to the onset of activation during development and uniformity of staining among individual cells within tissues. Overall, these mice will provide a powerful tool to researchers to study gene function in vascular development/disease by using cre/lox technology to direct smooth muscle-specific gene activation or inactivation in vivo.

Regulated expression of the BTEB2 transcription factor in vascular smooth muscle cells: analysis of developmental and pathological expression profiles shows implications as a predictive factor for restenosis.

BACKGROUND: We have previously shown BTEB2, a Krüppel-like zinc finger transcription factor, to regulate expression of the SMemb/NMHC-B gene, which has been implicated in phenotypic modulation of smooth muscle cells (SMCs). The present study was done to assess the developmental and pathological expression profiles of BTEB2 and to further evaluate the clinical relevance of BTEB2 expression in human coronary artery disease. METHODS AND RESULTS: Immunohistochemistry showed developmentally regulated expression of BTEB2 with abundant expression in fetal but not in adult aortic SMCs of humans and rabbits. In balloon-injured aortas, predominant expression of BTEB2 was seen in neointimal SMCs. Atherectomy specimens obtained from primary and restenotic lesions showed predominant expression of BTEB2 to stellate SMCs. The incidence of restenosis in primary lesions was significantly higher in lesions containing BTEB2-positive cells than in lesions without (55.6% versus 25.0%, P:=0.01). CONCLUSIONS: The present study shows that BTEB2 expression is developmentally and pathologically regulated. BTEB2 is preferentially expressed in dedifferentiated or activated SMCs. Examination of human coronary artery specimens suggests that primary lesions containing BTEB2-positive cells are associated with higher risk of restenosis than BTEB2-negative lesions. These results suggest that BTEB2 can serve as a molecular marker for phenotypic modulation of vascular SMCs.

Significance of the transcription factor KLF5 in cardiovascular remodeling.

Structural remodeling of the heart and blood vessels is an important pathologic process in the development of many cardiovascular diseases. However, transcriptional regulation of altered gene expression during cardiovascular remodeling is not well understood. We previously isolated KLF5/basic transcription element-binding (BTEB)2, a Krüppel-like factor, as a transcription factor that binds the promoter of the embryonic smooth muscle myosin heavy chain gene (SMemb). KLF5 activates many genes inducible during cardiovascular remodeling, such as platelet-derived growth factor (PDGF)-A/B, Egr-1, plasminogen activator inhibitor-1 (PAI-1), inducible nitric oxide synthase (iNOS), and vascular endothelial growth factor (VEGF) receptors. KLF5 is abundantly expressed in embryonic smooth muscles and is down-regulated with vascular development, but reinduced in proliferative neointimal smooth muscles in response to vascular injury. In KLF5 gene-targeted mice, homozygotes die at an early embryonic stage whereas heterozygotes are apparently normal. However, in response to external stress, arteries of heterozygotes exhibit diminished levels of smooth muscle and adventitial cell activation. Furthermore, angiotensin II-induced cardiac hypertrophy and fibrosis are attenuated in heterozygotes. KLF5 activities are regulated by many transcriptional regulators and nuclear receptors, such as retinoic acid receptor-alpha (RAR alpha), NF-kappaB, PPAR gamma, p300, and SET. Interestingly, RAR alpha agonist suppresses KLF5 and cardiovascular remodeling, whereas RAR alpha antagonist activates KLF5 and induces angiogenesis. These results indicate that KLF5 is an essential transcription factor in cardiovascular remodeling and a potential therapeutic target for cardiovascular disease.

A new type of familial central diabetes insipidus caused by a single base substitution in the neurophysin II coding region of the vasopressin gene.

We studied the genetic basis of familial neurohypophyseal diabetes insipidus in a Japanese family. The members had polyuria and a deficiency of plasma vasopressin (AVP). Polymerase chain reaction (PCR) amplified exons of the AVP-neurophysin-II gene were subcloned and sequenced. Exons 1 and 3 were normal, but nucleotide 1884 Guanine (G) in exon 2 was substituted with Thymine (T), which induced a substitution of glycine (Gly) for valine (Val). To examine the presence of this mutation in the affected subjects, we designed two mutated primers. One of them induced a new endonuclease restriction site in the PCR fragments from normal, and the other induced a new endonuclease restriction site from patients with the mutation. DNA fragments from two affected members of this family were amplified with this primer, and the PCR products were digested by endonuclease and resolved by electrophoresis. The results indicated that these subjects had both normal and mutant alleles, indicating that the mutation was heterozygous. We concluded that this mutation caused neurohypophyseal diabetes insipidus in this family.

Amitriptyline inhibits the G protein and K+ channel in the cloned thyroid cell line.

We have reported that thyroid K+ channel is activated by extracellular application of the thyroid-stimulating hormone (TSH) using single channel recording method performed on cloned normal rat thyroid cell (FRTL-5) membrane. Treatment of dibutyryladenosine cyclic monophosphate (Bt2 cAMP) also activated the TSH-dependent K+ channel. These findings indicate that the thyroid K+ channel is activated through the TSH-adenosine cyclic monophosphate (cAMP)-protein kinase A system. We examined the effects of amitriptyline on TSH-guanosine triphosphate binding protein (G protein)-adenylate cyclase-cAMP-K+ channel system in the cloned normal rat thyroid cell line FRTL-5. Amitriptyline inhibited the cAMP production induced by TSH. Amitriptyline also inhibited the cAMP production induced by cholera toxin, indicating that amitriptyline inhibited the thyroid G protein. Amitriptyline had no effect on TSH-receptor binding and cAMP production by forskolin (adenylate cyclase stimulator). Amitriptyline inhibited the K+ channel activation by cAMP, indicating that the suppressing mechanism is not the inhibition of TSH receptor or G protein but the direct suppression of K+ channel. It was concluded that amitriptyline inhibited the thyroid G protein and K+ channel.

Tonic block of the Na+ current in single atrial and ventricular guinea-pig myocytes, by a new antiarrhythmic drug, Ro 22-9194.

Ro 22-9194 reduced the Na+ current in the atrial myocytes as well as ventricular myocytes in a tonic block fashion. Ro 22-9194 had a higher affinity to the inactivated state Na+ channels (KdI = 3.3 microM in atrial myocytes, KdI = 10.3 microM in ventricular myocytes) than to those in the rested state (KdR = 91 microM in atrial myocytes, KdR = 180 microM in ventricular myocytes), which indicated that Ro 22-9194 had a higher affinity to the Na+ channels in atrial myocytes than in ventricular myocytes. Ro 22-9194 shifted the inactivation curve in the hyperpolarized direction in both atrial and ventricular myocytes. These findings suggest that Ro 22-9194 more strongly inhibited the Na+ channel of the atrial myocytes of the diseased hearts with the depolarized membranes potentials than the Na+ channels in ventricular myocytes.

Hematuria in patients with renal hypouricemia.

The characteristics of urate metabolism in renal hypouricemic patients with hematuria were studied to clarify the risk factors for hematuria in patients with renal hypouricemia. In 16 Japanese patients with isolated renal hypouricemia, urate metabolism was measured using the urate clearance study and the subtype of renal hypouricemia [defective presecretory reabsorption (Pre), defective postsecretory reabsorption (Post), enhanced tubular secretion (Secretion) and defective presecretory and postsecretory reabsorption (Pre&Post)] were determined by the pharmacological tests. Hematuria was seen in 7 out of the 16 patients (44%), all of whom were females (58%). Serum urate and urinary urate concentrations were significantly higher in the group with hematuria (Sur = 1.76 +/- 0.31 mg/dl and Uur/Ucr = 0.75 +/- 0.12: p<0.05) than in the group without hematuria (Sur = 1.44 +/- 0.46 mg/dl and Uur/Ucr = 0.56 +/- 0.04), although there was no difference in the urate excretion rate between the two groups. Hematuria was more likely to be accompanied by Post (75%) and Secretion (75%), which showed significantly higher urinary urate concentration (Uur/Ucr = 0.75 +/- 0.1 and 0.69 +/- 0.13, respectively) than by Pre (25%) and Pre&Post (0%), which showed lower urinary urate concentration (0.61 +/- 0.06 and 0.62 +/- 0.05, respectively). The risk factors for hematuria in patients with renal hypouricemia are the elevation of urinary urate concentration and the subtypes of Post and Secretion.

Renal handling of urate in a patient with familial juvenile gouty nephropathy.

We encountered a case of familial juvenile gouty nephropathy (FJGN) with an autosomal dominant transmission pattern. Hyperuricemia in the propositus was caused by renal underexcretion of urate although his erythrocyte purine enzyme was normal. A renal biopsy specimen from the propositus showed interstitial fibrosis with tubular atrophy. On pyrazinamide and probenecid tests, the tubular secretion of urate selectively decreased without changes in either presecretory or postsecretory reabsorption of urate when his renal function was normal. Probenecid increased the urinary urate excretion and Cur/Ccr. The serum urate concentration was poorly controlled by allopurinol. When his renal function deteriorated, the uricosuric effects of both probenecid and benzbromarone were attenuated. However, the combined administration of probenecid with allopurinol decreased the serum urate concentration. These data suggest that the tubular secretion of urate is selectively impaired in FJGN and at the stage of renal failure, the combination of an uricosuric agent with allopurinol might be effective in treating hyperuricemia in FJGN.

The smooth muscle myosin heavy chain gene exhibits smooth muscle subtype-selective modular regulation in vivo.

Previous studies in our laboratory demonstrated that the transgene consisting of the -4.2 to +11.6 kilobase (kb) region of the smooth muscle (SM) myosin heavy chain (MHC) gene was expressed in virtually all SM tissue types in vivo in transgenic mice and that the multiple CArG elements within this region were differentially required in SMC subtypes, implying that the SM-MHC gene was controlled by multiple transcriptional regulatory modules. To investigate this hypothesis, we analyzed specific regulatory regions within the SM-MHC -4.2 to +11.6 kb region by a combination of deletion analyses of various SM-MHC transgenes as well as by DNaseI hypersensitivity assays and in vivo footprinting in intact SMC tissues. The results showed that SM-MHC transgene expression depended on a large number of required regulatory modules that were widely spread over the -4.2 to +11.6 region. Moreover, the results revealed several unexpected novel features of regulation of the SM-MHC gene including: 1) unique combinations of regulatory modules were required for SM-MHC expression in different SMC-subtypes; 2) repressor modules as well as activator modules were both critical for SMC specificity of the gene; 3) certain modules were required in certain contexts but were dispensable in others within a given SMC-subtype (i.e. the net activity of the module was determined by interaction between modules not simply by the sum of module activities); and 4) we identified a highly conserved 200-base pair transcriptional regulatory module at +8 kb that was required in the large arteries but dispensable in the coronary arteries and airways in transgenic mice and contained multiple potential cis-elements that were occupied by nuclear proteins in the intact aorta based on in vivo footprinting. Taken together, the results suggest a model of complex modular control of expression of the SM-MHC gene that varies between SMC subtypes. Moreover, the studies establish the possibility of designing derivatives of the SM-MHC promoter that might be used for targeting gene expression to specific SMC subtypes in vivo.

Facilitation of beta-adrenoceptor-mediated slow channel responses by hypoxia in guinea pig ventricular myocardium.

The effects of hypoxia on the beta-adrenoceptor-mediated slow channel responses of guinea pig ventricular muscle in a potassium-rich (27 mM) solution containing Ba2+ were examined using microelectrode techniques. Isoproterenol produced a small depolarization of the resting membrane and also induced repetitive discharges of action potentials at higher concentrations, mainly due to a beta-adrenoceptor-mediated increase in the slow channel conductance. Two different threshold concentrations of isoproterenol, one for inducing depolarization and one for inducing automatic activity, were measured to assess myocardial responsiveness to catecholamines. During hypoxia, the electrically triggered slow upstroke action potentials of muscle were gradually depressed and catecholamine-induced membrane responses mediated by the beta-adrenoceptor/slow channel system were enhanced. The enhancement of the catecholamine effects was accelerated by acidosis and reversed by reoxygenation. Methyl xanthine-induced responses, similar to those induced by catecholamines, were also enhanced during hypoxia. It is suggested that not only changes of catecholamine-beta-adrenoceptor interaction, but also changes of intracellular metabolic processes, may be responsible, at least in part, for the enhancement of abnormal automatic activity mediated by the myocardial beta-adrenoceptor/slow channel system under hypoxic conditions.

Nucleotide sequence comparison of the mycobacterial dnaJ gene and PCR-restriction fragment length polymorphism analysis for identification of mycobacterial species.

We recently reported a genus-specific PCR for the mycobacterial dnaJ gene. In the present study, we have determined the nucleotide sequences of the dnaJ gene from 19 mycobacterial species (Mycobacterium tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti, M. marinum, M. kansasii, M. gastri, M. simiae, M. scrofulaceum, M. szulgai, M. gordonae, M. avium, M. intracellulare, M. xenopi, M. fortuitum, M. chelonae, M. hemophilum, and M. paratuberculosis). On the basis of the amplified dnaJ gene nucleotide sequences, we constructed a phylogenetic tree of the mycobacterial species by using the neighbor-joining method and unweighted pairwise grouping method of arithmetic average. We found that the phylogenetic relationship inferred within the slowly growing species was in good agreement with the traditional classification, with three major branches corresponding to Runyon's groups I, II, and III. An exception was M. simiae, which was phylogenetically closer to the cluster including members of Runyon's group III than to that of Runyon's group I. On the other hand, the rapid growers, such as M. fortuitum and M. chelonae, did not form a coherent line corresponding to Runyon's group IV, indicating that our phylogenetic analysis based on the dnaJ gene reflects the phenotypic characteristics such as pigmentation but not the growth rate. Finally, we revealed the species-specific restriction sites within the amplified dnaJ gene to differentiate most of the mycobacterial DNA by a combination of PCR with restriction fragment length polymorphism analysis.

Smooth muscle-specific expression of the smooth muscle myosin heavy chain gene in transgenic mice requires 5'-flanking and first intronic DNA sequence.

The smooth muscle myosin heavy chain (SM-MHC) gene encodes a major contractile protein whose expression exclusively marks the smooth muscle cell (SMC) lineage. To better understand smooth muscle differentiation at the transcriptional level, we have initiated studies to identify those DNA sequences critical for expression of the SM-MHC gene. Here we report the identification of an SM-MHC promoter-intronic DNA fragment that directs smooth muscle-specific expression in transgenic mice. Transgenic mice harboring an SM-MHC-lacZ reporter construct containing approximately 16 kb of the SM-MHC genomic region from -4.2 to + 11.6 kb (within the first intron) expressed the lacZ transgene in all smooth muscle tissue types. The inclusion of the intronic sequence was required for transgene expression, since 4.2 kb of the 5'-flanking region alone was not sufficient for expression. In the adult mouse, transgene expression was observed in both arterial and venous smooth muscle, in airway smooth muscle of the trachea and bronchi, and in the smooth muscle layers of all abdominal organs, including the stomach, intestine, ureters, and bladder. During development, transgene expression was first detected in airway SMCs at embryonic day 12.5 and in vascular and visceral SMC tissues by embryonic day 14.5. Of interest, expression of the SM-MHC transgene was markedly reduced or absent in some SMC tissues, including the pulmonary circulation. Moreover, the transgene exhibited a heterogeneous pattern between individual SMCs within a given tissue, suggesting the possibility of the existence of different SM-MHC gene regulatory programs between SMC subpopulations and/or of episodic rather than continuous expression of the SM-MHC gene. To our knowledge, results of these studies are the first to identify a promoter region that confers complete SMC specificity in vivo, thus providing a system with which to define SMC-specific transcriptional regulatory mechanisms and to design vectors for SMC-specific gene targeting.

Isolation of the embryonic form of smooth muscle myosin heavy chain (SMemb/NMHC-B) gene and characterization of its 5'-flanking region.

To examine the molecular mechanisms that regulate the expression of the SMemb/NMHC-B gene, a nonmuscle myosin heavy chain isoform predominantly expressed in fetal aorta, we have isolated and characterized the 5'-flanking region of the rabbit SMemb/NMHC-B gene. Transient transfection experiments demonstrated that 105 base pairs of 5'-flanking sequence was necessary to direct high level transcription in C2/2 cells, vascular smooth muscle cells derived from rabbit aorta. An essential cis-regulatory element was localized between -100 and -91 base pairs from the transcription start site based on the results that replacement mutagenesis within this region significantly reduced promoter activity. Sequence of this region is completely conserved between mouse and rabbit and fits no known DNA binding consensus. Gel mobility shift assays revealed that a specific DNA-protein complex was formed at this site with nuclear extracts from C2/2 cells, which can be competed by H-2Kb CCAAT box but not by Hsp70 CCAAT box or other CCAAT-containing sequences. We conclude that expression of the SMemb/NMHC-B gene is regulated through an interaction between a sequence element located at -100 and a distinct member of CCAAT-binding proteins.

Activation of Na(+)-H+ antiporter (NHE-1) gene expression during growth, hypertrophy and proliferation of the rabbit cardiovascular system.

The Na(+)-H+ antiporter is a unique transmembrane protein with multiple roles in cellular functions through intracellular alkalization. It participates in the regulation of intracellular pH, cell volume and intracellular signalling in response to various mitogenic stimuli. To clarify its role as a subcellular signal in cardiovascular remodeling like vascular hyperplasia or cardiac hypertrophy, we determined mRNA levels of the Na(+)-H+ antiporter isoform, NHE-1, in vascular smooth muscles and pressure-overloaded hearts in rabbits. The NHE-1 mRNA levels in rabbit aortas and hearts were developmentally regulated with high levels at embryonic and neonatal stages than in adults. In primary-cultured smooth muscle cells (SMC), the mRNA levels were increased during exponential growth, but decreased to initial levels at confluency. Growth of a mutant SMC line, C5, which is deficient in Na(+)-H+ antiporter activity, was markedly reduced in bicarbonate-free medium. However, when the activity was restored by transfecting cells with a full-length NHE-1 cDNA in an expression vector, the growth rate of C5 was accelerated again. After balloon injury to the vascular wall, the NHE-1 mRNA levels of the injured arteries were also increased, suggesting that Na(+)-H+ antiporter contributes to the network of the growth promoting systems in smooth muscle cells in vivo. Pressure-overload on the ventricle increased the NHE-1 mRNA levels in hearts approximately two-fold of sham-operated rabbits after 3 days and remained for at least two weeks (P < 0.05). We further demonstrated that 3-methylsulfonyl-4-piperidino-benzoyl guanidine mesylate (Hoe 694), a potent antagonist of Na(+)-H+ antiporter, partially inhibited stretch-induced activation of mitogen-activated kinase (MAP kinase) in the cultured cardiomyocytes. From these results, we conclude that activation of the Na(+)-H+ antiporter and its gene expression is involved in molecular mechanisms of both cardiac hypertrophy and vascular smooth muscle cell proliferation, indicating a potential target in developing new therapeutics for cardiovascular diseases.

Redifferentiation of smooth muscle cells after coronary angioplasty determined via myosin heavy chain expression.

BACKGROUND: The pathophysiology of phenotypic modulation of smooth muscle cells (SMCs) involved in restenosis after angioplasty is not well understood. Smooth muscle myosin heavy chain (SM MHC) isoforms (SM1 and SM2) are specific markers for SMC differentiation. In particular, SM2 is useful as a marker of mature SMCs. SMemb is a nonmuscle myosin heavy chain (NM MHC) whose expression is upregulated in immature or activated SMC. METHODS AND RESULTS: To determine SMC phenotypes in neointimal tissues after percutaneous transluminal coronary angioplasty (PTCA), we performed immunohistochemistry on human coronary arteries with antibodies against alpha-SM actin, SM1, SM2, and SMemb. Tissues were obtained from six autopsied patients and from atherectomy specimens from 16 patients who had undergone PTCA. Medial SMCs were positive for alpha-actin, SM1, and SM2. Expression of SM1 and SM2 in the neointima varied with the time after intervention, whereas alpha-actin was constitutively expressed in all cases studied. Neointimal cells at 16 and 20 days after PTCA contained alpha-actin but little or no SM1 or SM2, indicating that these cells modulated their phenotype to the immature state. Neointimal SMCs recovered SM MHC expression, first SM1 and then SM2, by 6 months after PTCA. Increased expression of SMemb was found in the neointima but without apparent relationship to the time after PTCA. CONCLUSIONS: Neointimal SMCs show features of an undifferentiated state, indicated by altered expression of SM MHC, and undergo redifferentiation in a time-dependent manner. The expression of SM MHC isoforms provides insight into the biology of healing after angioplasty and furnishes useful tools for the understanding of the roles of differentiation and phenotypic modulation of SMCs in human vascular lesions.