Yu-Shien Ko

Predictors of Systemic Embolism in Patients with Mitral Stenosis: A Prospective Study

Systemic, especially cerebral, embolism is one of the major causes of illness and death in patients with mitral stenosis [1-5]. Identification of risk factors for embolism may improve the strategies for preventing this event. However, most large studies addressing risk predictors have been retrospective [1, 2, 6-9]. We sought to reappraise this issue in a large, prospective study. Methods Patients Eligible patients were consecutive adults (age 15 years) with mitral stenosis (mitral valve area 2 cm2 according to echocardiographic planimetry) who presented to a university-affiliated medical institution from April 1987 to December 1994. We excluded patients with infective endocarditis and those who were in critical condition because of systemic embolism and died during hospitalization. End Point The study end point was the occurrence of new systemic embolism during follow-up. The diagnosis of systemic embolism was based on symptoms and signs (sudden onset of peripheral arterial ischemic [for example, sudden flank pain with hematuria, abdominal pain with gastrointestinal bleeding, or leg pain with pulse deficit] or neurologic manifestations without prodromes) and on findings from computed tomography, angiography, and surgery. We did not attempt to detect silent emboli. Clinical Variables We assessed nine clinical variables (Table 1): age at enrollment; sex; presence or absence of previous systemic embolism, atrial fibrillation, hypertension, and New York Heart Association class III or IV congestive heart failure; and therapy with anticoagulants, percutaneous balloon mitral commissurotomy, or valvular surgery. Patients were regularly followed at outpatient clinics. Table 1. Clinical and Echocardiographic Variables in 534 Patients with Mitral Stenosis* Echocardiographic Method and Variables Standard transthoracic echocardiography was done at enrollment in all patients by using a Hewlett-Packard 7340, Sonos 1000, or Sonos 1500 echocardiographic system (Hewlett-Packard, Palo Alto, California) interfaced with both 2.5-MHz and 5.0-MHz transducers. Biplane or omniplane transesophageal echocardiography using a 5.0-MHz transducer was also performed in a subgroup of consecutive patients who entered the study from September 1991 to October 1992. Ten echocardiographic variables were examined (Table 1). Mitral valve area was measured by planimetry from two-dimensional echocardiography. When two-dimensional echocardiography of the mitral orifice yielded unsatisfactory results, we used the pressure half-time (T1/2) method (mitral valve area [cm2] = 220/T1/2 ms) [10-12]. We did not use the pressure half-time method when the mitral orifice could be clearly defined by two-dimensional echocardiography because pressure half-time is influenced by many factors other than mitral valve area [13, 14]. Other echocardiographic variables were left atrial diameter at end systole; presence or absence of a left atrial thrombus [15] or left atrial smoky echoes on transthoracic or transesophageal echocardiography [16-18]; presence or absence of impaired left ventricular systolic performance; and presence or absence of significant (moderate or severe) aortic stenosis, aortic regurgitation, mitral regurgitation, tricuspid regurgitation, or pulmonic regurgitation. The degrees of these valvular lesions were semiquantified by using a continuity equation (for aortic stenosis) or color Doppler imaging (for various regurgitations), as described elsewhere [19, 20]. Briefly, significant aortic stenosis refers to an aortic valve area of 1.2 cm2 or less determined by the continuity Equation method RF 19*; significant mitral or tricuspid regurgitation refers to a ratio of regurgitant jet area to left or right atrial area of 20% or more; and significant aortic or pulmonic regurgitation refers to a ratio of jet width to ventricular outflow tract diameter of 25% or more [20]. Left atrial diameter at end systole was measured from an M-mode echocardiogram recorded in parasternal long-axis view. The measurement was made according to the recommendations of the American Society of Echocardiography [21]. To detect left atrial smoky echoes, we used a 5-MHz transducer during transthoracic and transesophageal echocardiography because a 5-MHz transducer is more sensitive than a 2.5-MHz transducer [16]. We chose the term smoky echoes instead of spontaneous echocardiographic contrast (a term frequently used in other studies [6, 7]) because some patients with severe tricuspid regurgitation or right heart failure had bright moving spots (originating from microbubbles) in the venae cavae or hepatic veins that were identical to those seen during contrast echocardiography. Thus, we reserve spontaneous echocardiographic contrast for that echocardiographic pattern and use smoky echoes for the finer, lighter whorling echoes (originating from aggregated erythrocytes) [16-18] that appeared in the left atrium in patients with severe mitral stenosis. Statistical Analysis For each clinical and echocardiographic measure, the log-rank statistic was used to determine whether the overall pattern of the time to development of systemic embolism (embolism-free time) varied among levels of the measure. Mean embolism-free time was estimated by using a nonparametric method that considers censoring [22]. Cox regression was used to examine the significance of the clinical and echocardiographic variables in predicting embolism-free time for patients in sinus rhythm and patients in atrial fibrillation. All analyses were performed by using BMDP Dynamic Release 7.0 [23]. Results Five hundred thirty-four patients were followed for a mean (SD) of 36.9 22.5 months. Of these, 257 patients (48.1%) received anticoagulants throughout the follow-up period. The indications for anticoagulation were the presence of a left atrial thrombus, atrial fibrillation, or a history of systemic embolism; patient compliance with therapy; and lack of risk factors for bleeding. The relatively low percentage of patients receiving anticoagulants in this series was due to minimal patient compliance. During the follow-up period, 60 patients (11.2%) developed a systemic embolism. When Cox regression was performed, significant interaction was found between atrial fibrillation and age, percutaneous balloon mitral commissurotomy, mitral valve area, previous systemic embolism, left atrial thrombus, and anticoagulation. In other words, the significance of these variables depended to some extent on whether the patient was in atrial fibrillation or sinus rhythm. We therefore performed subgroup analyses. Subgroup Analyses Of the 132 patients in sinus rhythm, 12 (9.1%) developed systemic embolism during follow-up. Age (P < 0.001), percutaneous balloon mitral commissurotomy (P = 0.02), and mitral valve area (P = 0.02) were significant predictors in the log-rank analysis (Table 2). Results of the Cox regression showed that age (relative risk [RR], 1.12 [95% CI, 1.04 to 1.21]), left atrial thrombus (RR, 37.1 [CI, 2.82 to 487.8]), mitral valve area (RR, 16.9 [CI, 1.53 to 187.0]), and significant aortic regurgitation (RR, 22.4 [CI, 2.72 to 184.8]) were significant predictors of new systemic embolism (Table 3). No interactions were found among these variables. However, mitral valve area became a nonsignificant predictor (P = 0.12) when patients with percutaneous balloon mitral commissurotomy were excluded from the analysis. Table 2. Subgroup Univariate Analysis of Correlates of Systemic Embolism in Patients with Mitral Stenosis* Table 3. Cox Regression Analysis of Predictors of Systemic Embolism in Patients with Mitral Stenosis* Of the 402 patients in atrial fibrillation, 48 (11.9%) developed systemic embolism. Age (P = 0.01), previous embolism (P = 0.001), and percutaneous balloon mitral commissurotomy (P = 0.003) were significant predictors in the univariate analysis (Table 2). In the multivariate analysis, however, only previous embolism (RR, 3.11 [CI, 1.66 to 5.85]) and percutaneous balloon mitral commissurotomy (RR, 0.37 [CI, 0.18 to 0.79]) remained significant predictors of embolism-free time (Table 3). Again, no interactions were found between these two variables. A subgroup analysis of the 164 patients who underwent baseline transesophageal echocardiography revealed no other significant predictors. Discussion Factors Correlated with Systemic Embolization in Patients with Mitral Stenosis Our prospective study revealed that for patients in sinus rhythm, embolization was related to age, mitral valve area, the presence of a left atrial thrombus, and significant aortic regurgitation. For patients in atrial fibrillation, the significant factors were previous embolism and percutaneous balloon mitral commissurotomy (Table 3). Other retrospective studies have shown that atrial fibrillation [1, 2, 8], age [1, 2, 8], and previous embolism [3] correlate with increased incidence of systemic embolism in patients with mitral stenosis and that age is closely related to the prevalence of atrial fibrillation [24] and to a history of embolization [1, 2, 8]. Several studies have shown that anticoagulation reduces the incidence of systemic embolism in patients with mitral stenosis and atrial fibrillation [25-27]. To the best of our knowledge, however, our study is the first to show that the presence of a left atrial thrombus and significant aortic regurgitation are positive predictors and that percutaneous balloon mitral commissurotomy seems to be a negative predictor. Left Atrial Thrombus and Systemic Embolism in Patients with Mitral Stenosis Who Are in Sinus Rhythm Dislodgement of a left atrial thrombus in patients with mitral stenosis has been thought to lead to systemic embolism. Although a correlation between left atrial thrombus and systemic thrombus would be expected, previous studies have not confirmed such a correlation. We found that the presence of a left atrial thrombus was a positive predictor (RR, 37.1 [CI, 2.82 to 487.8]) for patients in sinus

The Gln–Arg 191 polymorphism of the human paraoxonase gene is not associated with the risk of coronary artery disease among Chinese in Taiwan

Paraoxonase (PON1) is a high density lipoprotein-associated enzyme capable of hydrolyzing lipid peroxides, and thus, might protect lipoproteins from oxidation. A common polymorphism due to an amino acid substitution (Gln-Arg) at codon 191 is considered to be a major determinant of variation in serum PON1 activity. Recent studies have suggested that the PON1-191 polymorphism is an independent risk factor for coronary atherosclerosis in patients with or without diabetes mellitus. The association of PON1-191 polymorphism genotypes and coronary artery disease (CAD) among Chinese subjects in Taiwan was examined. The genotype of 218 angiographically documented CAD patients and the same number of age- and sex-matched control subjects was determined. Genotypes AA, AB and BB were present in 25 (11%), 102 (47%) and 91 (42%) of control subjects, respectively, and in 30 (14%), 96 (44%) and 92 (42%) of CAD patients, respectively (chi2 = 0.57, P = 0.75 between groups). The frequency of the A allele was 0.36 for the control group and 0.35 for CAD patients (P = 0.94). No significant differences in the PON1-191 genotype frequencies could be found between groups when multivariate logistic regression analysis was performed, or different subgroups of age, sex or risk factors were analyzed. Among control subjects, there was also no significant difference between genotypes of the PON1-191 polymorphism and various clinical and lipid variables. In conclusion, our data suggest that there is no association between the Gln-Arg 191 polymorphism of the human PON1 gene and CAD among Chinese subjects in Taiwan.

Age-related alteration of gap junction distribution and connexin expression in rat aortic endothelium.

We investigated endothelial gap junctions and their three component connexins, connexin37 (Cx37), Cx40, and Cx43, during growth and senescence in rat aorta by en face immunoconfocal microscopy and electron microscopy. Gap junction spots labeled by specific antisera against Cx37, Cx40, and Cx43 were quantified at 1 day, 7 days, 28 days, 16 months, and ≥20 months of age, and the relationship between the connexins was examined by co-localization analysis. At birth, all three connexins were abundantly expressed; the number and total area of connexin spots then declined within 1 week (p < 0.05 for each connexin). From 1 week, each connexin showed a distinct temporal expression pattern. Whereas Cx43 signal decreased progressively, Cx37 signal fluctuated in a downward trend. By contrast, Cx40 maintained an abundant level until ≥20 months of age (≥20 months vs 28 days, p < 0.05 for number and total connexin signal area). These patterns were associated with changes in endothelial cell morphology. Double-label analysis showed that the extent of co-localization of connexins to the same gap junctional spot was age-dependent [>70% at birth and 28 days old; <70% at later stages (p < 0.05)]. We conclude that expression of the three connexins in aortic endothelium is age-related, implying specific intercellular communication requirements during different stages after birth.

Multiple Connexin Expression in Regenerating Arterial Endothelial Gap Junctions

Endothelial cells form gap junctions that, according to vessel type, may be composed of up to 3 types of connexin, connexin37, connexin40, and connexin43. Although changes in connexin expression have been linked to growth and injury in cultured endothelial cells, information on connexin expression in regenerating endothelium in situ is lacking. We investigated gap junction distribution and expression of all 3 endothelial connexins during healing in rat carotid artery after denudation injury. En face viewing of the vascular luminal surface by means of immunoconfocal microscopy was used to examine the spatial and temporal expression pattern of the endothelial connexins. Gap junction spots labeled by specific antisera against connexin37, connexin40, and connexin43 were quantified 7, 14, and 28 days after injury, and the relations among the connexins were examined by using colocalization analysis. Complementary electron microscopy was also conducted. After injury, the regenerating endothelium initially expressed small, sparse gap junctions, the numbers of which progressively increased to values equivalent to those of controls. Although connexin40 gap-junctional spot size and area returned to uninjured levels by 28 days after injury, connexin37 and connexin43 spot size and area exceeded those of the uninjured artery (P<0.05). Double-label analysis showed that even though colocalization of connexins to the same gap-junctional spot is a common feature, the extent of colocalization was time dependent (>80% in the intact artery at postinjury day 28 and <70% at postinjury days 7 and 14, P<0.01). We conclude that distinct alterations in expression of the 3 connexins are associated with regeneration of the arterial endothelium in situ, implying different intercellular communication requirements during the various phases of the healing process.

Reduced expression of endothelial connexin37 and connexin40 in hyperlipidemic mice: recovery of connexin37 after 7-day simvastatin treatment.

Objective—We sought to clarify the response of endothelial connexins to hyperlipidemia and lipid-lowering therapy. Methods and Results—Aortic endothelial gap junctions were analyzed by en face immunoconfocal microscopy and electron microscopy in C57BL/6 mice subjected to the following regimens: (1) normal chow (NC) for 3 months (3 mo), (2) NC for 9 mo, (3) NC for 3 mo, followed by a cholesterol-enriched diet (CED) for 6 mo, (4) NC for 3 mo and CED for 6 mo, with simvastatin in the final week, and (5) (in apoprotein E [apoE]-deficient mice) NC and examined at 3 mo and 7 to 9 mo. In wild-type mice, connexin37 (Cx37) and Cx40 were markedly downregulated in the CED-fed animals compared with those fed NC (CED vs 9-mo NC, 77% reduction in Cx37 and 65% reduction in Cx40; both P <0.01). After simvastatin treatment, Cx40 remained depressed, but Cx37 recovered to 94% of the level found in non–cholesterol-fed animals (P <0.01). Electron microscopy demonstrated that gap junctions were smaller in animals fed the CED compared with those given simvastatin and with controls fed NC (P <0.01). Endothelial connexins were rare in the atherosclerotic plaques of apoE-deficient mice. Conclusions—Mouse aortic endothelial gap junctions and connexins are downregulated during long-term hyperlipidemia. Short-term treatment with simvastatin leads to recovery of Cx37 expression but not Cx40 expression.

Angiotensinogen and angiotensin-I converting enzyme gene polymorphisms and the risk of coronary artery disease in Chinese

Abstract The homozygous deletion allele (DD) of the angiotensin-I converting enzyme (ACE) gene and the T235 homozygote of the angiotensinogen (AGT) gene have been reported to be correlated with an increased prevalence of coronary artery disease (CAD) and myocardial infarction (MI). The importance of the DD genotype and T235 homozygote as genetic risk factors for CAD in Chinese remains uncertain. This study included 426 patients who underwent coronary angiography and 180 healthy subjects without clinical evidence of CAD. Coronary angiography identified 268 patients with CAD (CAD group) and 158 patients without CAD. The healthy subjects and patients without angiographic evidence of CAD constituted the control group. Three polymorphisms were studied: an insertion/deletion (I/D) polymorphism of the ACE gene and the T174 M and M235T polymorphisms of the AGT gene. No association was found between any of the three studied polymorphisms and the risk of CAD or MI in Chinese using univariate or multivariate analysis. In multivariate analysis, the relative risks were 1.20 (95% confidence interval = 0.91–1.61, P = 0.20) for the DD genotype, 1.05 (95% CI = 0.82–1.35, P = 0.69) for the T174 homozygote, and 1.19 (95% CI = 0.91–1.55, P = 0.20) for the T235 homozygote. Similarly, no significant difference was found in the frequencies of the DD genotype and the T174 and T235 homozygotes between the control group, the CAD group, the non-MI group, and the MI group when analyzed according to sex, age, or degree of risk. Our data suggest that neither the DD genotype of the ACE I/D polymorphism nor the T174 and T235 homozygotes of the AGT gene confer significant risk for CAD or MI in Chinese.

Connexin Make-up of Endothelial Gap Junctions in the Rat Pulmonary Artery as Revealed by Immunoconfocal Microscopy and Triple-label Immunogold Electron Microscopy

Integration of vascular endothelial function relies on multiple signaling mechanisms, including direct cell-cell communication through gap junctions. Gap junction proteins expressed in the endothelium include connexin37, connexin40, and connexin43. To investigate whether individual endothelial cells in vivo express all three connexin types and, if so, whether multiple connexins are assembled into the same gap junction plaque, we used affinity-purified connexin-specific antibodies raised in three different species to permit multiple-label immunoconfocal and immunoelectron microscopy in the rat main pulmonary artery. Immunoconfocal microscopy showed a high incidence of co-localization between connexin43 and connexin40, but lower incidences of co-localization between connexin37 and connexin40 or connexin43. Immunoelectron microscopy revealed that 83% of gap junction profiles contained all three connexins, with the proportion of connexin40 labeling being significantly higher than that of connexin37 or connexin43. The presence of three different connexin types of distinct properties in vitro provides potential for complex regulation and functional differentiation of endothelial intercellular communication properties in vivo.

Three-Dimensional Reconstruction of the Rabbit Atrioventricular Conduction Axis by Combining Histological, Desmin, and Connexin Mapping Data

Background—The 3D structure of the atrioventricular conduction axis incorporating detailed cellular and molecular composition, especially that relating to gap-junctional proteins, is still unclear, impeding mechanistic understanding of cardiac rhythmic disorders. Methods and Results—A 3D model of the rabbit atrioventricular conduction axis was reconstructed by combining histological and immunofluorescence staining on serial sections. The exact cellular boundaries, especially those between transitional cells and atrial myocardium, were demarcated by a dense and irregular desmin-labeling pattern in conductive myocardium. The model demonstrates that the atrioventricular conduction axis is segregated into 2 connecting compartments, 1 predominantly expressing connexin45 (compact node and transitional cells) and the other predominantly coexpressing connexin43 and connexin45 (His bundle, lower nodal cells, and posterior nodal extension). The transitional zone shows unique features of spatial complexity, including a bridging bilayer structure (a deep transitional zone connecting with a superficial atrial-transitional overlay) and asymmetrical continuity (wider atrial-transitional interfaces and shorter atrial-axial distances in the hisian portion than in the ostial portion). In the latter compartment, the His bundle, lower nodal cells, and posterior nodal extension form a continual axis and longitudinal transitional-axial interface. Conclusions—Key findings of the present study are the demonstration of a distinct anatomical border between transitional and atrial cells, connection between transitional cells and both lower nodal cells and posterior nodal extension, and distinctive connexin expression patterns in different compartments of the rabbit atrioventricular conduction axis. These features, synthesized in a novel 3D model, provide a structural framework for the interpretation of nodal function.

Interaction between obesity and genetic polymorphisms in the apolipoprotein CIII gene and lipoprotein lipase gene on the risk of hypertriglyceridemia in Chinese

Abstract To understand the effects of the interaction between genetic polymorphisms and obesity on the risk of hypertriglyceridemia (HTG), two polymorphisms, an SstI polymorphism on the apolipoprotein CIII gene and a HindIII polymorphism on the lipoprotein lipase gene, were analyzed in 339 Chinese subjects with (82 cases in the HTG group) or without HTG (257 cases in the control group). Our data revealed that the frequencies of obesity, the SstI minor allele (S2), and the HindIII major allele (H+) in the HTG group were significantly higher than in the control group. Subgroup analysis revealed that the association between these two polymorphisms and HTG occurred predominantly in nonobese subjects and in subjects with the less hypertriglyceridemic genotype of another polymorphism. Multivariate logistic regression analysis showed that all three risk factors (obesity, S2-containing chromosome, and H+ homozygosity) were associated with HTG, and an interaction was found between obesity and H+ homozygosity for the occurrence of HTG. The risk of HTG increased significantly with combinations of risk factors. Subjects can be divided into low or high risk groups for HTG using such combinations. These results provide evidence of interaction between obesity and the HindIII polymorphism of the lipoprotein lipase gene on the risk of HTG.

Differential Expression of Connexin43 and Desmin Defines Two Subpopulations of Medial Smooth Muscle Cells in the Human Internal Mammary Artery

Upregulation of connexin43-gap junctions is associated with transition of contractile vascular smooth muscle cells (SMCs) to the synthetic state. To determine whether phenotypically distinct subpopulations of medial SMCs differentially express connexin43, we investigated the human distal internal mammary artery, a structurally heterogeneous vessel with features ranging from elastic to elastomuscular to muscular. Immunoconfocal microscopy combined with quantitative analysis and complemented by in situ hybridization showed that SMCs in the elastic medial regions expressed high levels of connexin43 but low levels of desmin, whereas those of muscular medial regions expressed low levels of connexin43 but high levels of desmin. Ultrastructurally, SMCs of both regions were of the contractile phenotype, but the former cells were irregular in shape with relatively prominent synthetic organelles whereas the latter were spindle shaped with fewer synthetic organelles. Vimentin, smooth muscle alpha-actin, calponin, h-caldesmon, and myosin heavy chains (SM1 and SM2) were equally highly expressed by most cells in both subpopulations. The connexin43/desmin expression pattern of SMCs in regions of intimal thickening resembled those of elastic medial regions. These findings refine the view suggested from previous studies that high levels of connexin43 expression are associated with SMCs of a less contractile/more synthetic phenotype. In the internal mammary artery, the 2 subpopulations of SMCs with markedly different connexin43 expression levels both represent a differentiated contractile phenotype, but the subpopulation showing high levels of connexin43-gap junctions is characterized by low levels of desmin and structural features that reflect a more synthetic tendency.