Impact of cardiac transplantation on molecular pathology of ET-1, VEGF-C, and mitochondrial metabolism and morphology in dilated versus ischemic cardiomyopathic patients.

Little is known about the long-term impact of cardiac transplantation on activity and modifications of endothelin (ET)-1 system, vascular endothelial growth factor (VEGF), and mitochondrial metabolism and morphology in patients with ischemic cardiomyopathy (ICM) versus dilated cardiomyopathy (DCM). Messenger RNA (mRNA) expression levels of ET-1, endothelin converting enzyme (ECE)-1, VEGF-C, carnitine palmitoyltransferase (CPT)-1, and carnitine acetyltransferase (CARAT), as well as the number of normal, edematous, and degenerated mitochondria were assessed in left ventricular biopsies of 21 patients with DCM and 20 with ICM (New York Heart Association class III-IV) before and up to 3 months after cardiac transplantation. Cardiac samples of donated, nonfailing hearts served as controls (n=10). In cardiac biopsies of both ICM and DCM patients, ET-1, VEGF-C, CPT-1, and CARAT mRNA were up-regulated, whereas ECE-1 mRNA was down-regulated (P<0.05). Degenerated mitochondria had the highest number in both groups, followed by normal and edematous mitochondria. After cardiac transplantation, in ICM patients impaired gene expression levels decreased to, or below, normal levels, and the number of normal mitochondria increased (P<0.05). In implanted hearts of DCM patients, however, up-regulated ET-1 transcript levels persisted and the number of normal mitochondria decreased, whereas the number of degenerated mitochondria increased (P<0.05), and edematous mitochondria remained unchanged in number. These results show that cardiac transplantation corrects the impaired hemodynamic and echocardiographic parameters in both groups, whereas in DCM, the molecular pathology of ET-1 system and mitochondria persists. Therefore, it is more likely that these changes are the cause rather than a consequence of DCM.

Selective downregulation of VEGF-A(165), VEGF-R(1), and decreased capillary density in patients with dilative but not ischemic cardiomyopathy.

Cardiomyopathy (CM) comprises a heterogeneous group of diseases, including ischemic (ICM) and dilative (DCM) forms. The pathogenesis of primary DCM is not clearly understood. Recent studies in mice show that vascular endothelial growth factor (VEGF) is involved in ICM. Whether VEGF plays a role in human CM is unknown. We examined the mRNA and protein expression of VEGF and its receptors in hearts of patients with end-stage DCM and ICM and in healthy individuals using real-time polymerase chain reaction and Western blotting. Number of capillaries, area of myocytes, and collagen were calculated in cardiac biopsies using transmission electron microscopy. In DCM, except for VEGF-C, mRNA transcript levels of VEGF-A(165), VEGF-A(189), and VEGF-B and the protein level of VEGF-A and VEGF-R(1) were downregulated compared with controls (P:<0.05). However, in ICM, mRNA transcript levels of VEGF isoforms and protein levels of VEGF-C were upregulated. The vascular density was decreased in DCM but increased in ICM compared with controls (P:<0. 05). Muscular hypertrophy was not different for ICM and DCM, although DCM had more collagen (P:<0.05). Blunted VEGF-A and VEGF-R(1) protein expression and downregulated mRNA of the predominant isoform of VEGF-A, VEGF-A(165), to our knowledge shown here for the first time, provide evidence that the VEGF-A defect in DCM is located upstream. Whether downregulation of certain VEGF isoforms in DCM is a cause or consequence of this disorder remains unclear, although upregulated VEGF levels in ICM are most likely the result of ischemia.

Sphincters of canine hepatic sublobular veins respond to endothelin-1 and 3.

The dog has been used repeatedly as a model in liver transplantation research. The microcirculation and its regulatory mechanisms play a crucial role during ischemia and reperfusion. Little is known about the role of venous sphincters in regulating blood flow in the dog liver. Hence, we performed this study to elucidate their potential role in regulating local blood flow. In 14 dogs mean systemic (MSP) and mean portal venous pressure (MPP) were measured. Light and electron microscopy (scanning and transmission) of tissue sections and vascular corrosion casts were used to elucidate the microvascular morphology. Immunocytochemistry was applied to identify smooth muscle cells and the innervation of venous sphincters. Endothelins 1 and 3 were injected to find whether the hepatic venous sphincters are sensitive to these vasoactive agents. Tufts of smooth muscle cells were found in the sublobular veins (SLV; 100 to 250 microm in diameter), that reduced the luminal diameters of veins by 34%. Nerve endings were not observed close to these venous sphincters. The MSP and MPP were 75.3+/-2.4 mmHg and 8.9+/-0.95 mmHg, respectively. Treatment with 1.0 microg/kg of endothelin-1 (ET-1) significantly increased the MSP, the MPP and the percentage of focal venous sphincter contraction by 39% (105+/-4.7 mmHg), 43% (12.8+/-1.7 mmHg) and 57% (53.5+/-4.7), respectively (P <0.01). Treatment with ET-3 caused a significant (P <0.01) decrease in the MSP, the MPP and the percentage of sphincter contraction by 19% (61.0+/-2.2 mmHg), 39% (5.8+/-2.9 mmHg) and 38% (20.9%+/-3.15). Sinusoids did not contain sphincters. Hepatic arterioles and central veins were not affected by ET-treatment. The contraction of SLV sphincters correlated with increases in MPP (r=0.81, P <0.01) and was related to the MSP (r=0.67, P <0.01). These data show that the smooth muscle sphincters in SLV of the dog liver are involved in the local regulation of blood flow and that these sphincters are stimulated by non-neurogenic mechanisms. These sphincters contract in response to ET-1 and relax in response to ET-3. Since ET-1 is released during and/or causes inflammation, e.g., during ischemia and reperfusion, its antagonists might be of benefit during transplantation reperfusion of liver.

Endothelin-1 causes luminal constrictions in rat cochlear veins.

Serum levels of the vasoconstrictor endothelin-1 (ET-1) increase in ischemia and systemic hypertension. We examined the effects of ET-1 on the cochlear microvasculature. Blood vessels were cast with methacrylate in adult male Wistar Kyoto rats, 10 min after intravenous injection of ET-1 (1.0 microg/kg); control animals received saline. Systemic blood pressure was recorded continuously. ET-1 increased the average systolic pressure by 18% and average diastolic pressure by 22% (P < 0.01). Scanning electron microscopy of cast vessels showed multiple circumscribed luminal constrictions on: (1) postcapillary venules; (2) collecting veins; (3) where collecting veins merged with the spiral modiolar vein; (4) on the spiral modiolar vein itself. Circumscribed constrictions in arteries were not observed. In ET-1 injected animals focal contractions of collecting veins reduced luminal width by 13.4% +/- 2.9 (P < 0.01). In control rats, constrictions on venous casts were minimal and constrictions on arteries were not observed. The present study shows that ET-1 is involved in local control of cochlear blood flow in that it focally contracts cochlear veins. It is suggested that this might be due to the high affinity of ET-1 receptors and/or the large number of ET-1 receptors on contractile cells in venous walls.

Morphologic sites for regulating blood flow in the exocrine pancreas.

The exocrine pancreas has a lobular structure and an intricate capillary network supplies the lobules. Casts of these capillaries are either straight and of constant width, provided with many shallow crests, or undulating and of varying diameter, provided with bulges and deeper constrictions. The mean capillary cast diameter is 6.32 microns (SD 0.53) and 3.91 microns (SD 0.84) at constriction sites. The first type corresponds to non-fenestrated capillaries, makes 24% of capillaries and is more frequently provided with pericytes (2.7 +/- 0.9 pericytes per capillary profile). The second type corresponds to fenestrated capillaries, comprises 76% of the capillaries and is less frequently provided with pericytes (1.5 +/- 0.6 pericytes per capillary profile). The endothelial cells of capillaries regularly form intermediate junctions and microvilli and contain microtubuli and cytoplasmic filaments. Intravital observations show that capillaries are capable of contracting and narrowing the capillary lumen. This contractility is accomplished by endothelial cells both at and apart from their nuclear regions while pericytes never contracted spontaneously during our in vivo observations. The capillary diameters estimated by intravital measurements, 3.53 microns (SD 1.05), are similar to cast measurements but differ at constricted segments from cast measurements. Flow reduction shows more variability in smaller capillaries and the flow is more reduced in capillaries of 5 microns diameter to about 40% of open capillaries vs. 68% in capillaries with 7.5 microns diameter. Veins are either provided with smooth muscle sphincters or with valves. These results indicate that corrosion casting accurately shows the geometry of capillaries. However, where the capillaries are drastically constricted, they might not be filled and therefore may be underestimated during measurements. Since none of the intravital luminal constrictions are small enough to reduce flow (smaller than 1 micron luminal diameter) and because many constrictions are effective to reduce flow, we conclude that capillaries of the exocrine pancreas are always capable of maintaining continuous blood flow yet can influence blood perfusion. The presence of venous valves in association with venous sphincters constitutes a new situation concerning blood drainage regulation in the exocrine pancreas.

Closure of ruptured coronary sinus by a pericardial patch.

We report on a case of coronary sinus rupture that happened during placement of a cardioplegia balloon catheter and its subsequent repair. First, the defect was oversewn, however, not successfully. Under cardiopulmonary bypass and cardioplegic arrest, a pericardial patch was used to reconstruct the coronary sinus. Six months after the operation, blood drained during diastole into the right atrium but the flow was partially reversed during systole.

Spontaneously hypertensive rats develop pulmonary hypertension and hypertrophy of pulmonary venous sphincters.

This study explored the spontaneously hypertensive rat as an animal model of pulmonary hypertension and sought to identify anatomic changes in its pulmonary microvasculature, especially focal constrictions of pulmonary veins (sphincters). The average systemic and pulmonary artery blood pressures were 172/139 (+/- 9/9) and 36/14 (+/- 4/3), respectively, for spontaneously hypertensive Wistar Kyoto rats (SHR), and 134/83 (+/- 8/2) and 20/10 (+/- 2/2) for normotensive Wistar Kyoto rats (WKY) (P < 0.01 for both). Light microscopy of the lungs in SHR showed muscularization of both arteries and veins, but this was more pronounced in the small pulmonary veins. Perivascular edema was also present. There were 20 (+/- 4) leukocytes per 100 microns of capillary length in SHR and 9 (+/- 2) in WKY (P < 0.001). Transmission electron microscopy showed focal venous smooth muscle was greater in SHR than in WKY. Scanning electron microscopy of vascular casts showed the average maximal focal venous contraction (sphincter) was 54% (+/- 10) of its diameter in SHR, but was only 6% (+/- 4) in WKY (P < 0.01). Arterial contraction occurred in the hypertensive rats as bourglass narrowings of the casts, but was less conspicuous than venous constrictions. The mean alveolar capillary diameter was 8.1 microns (+/- 1.6) in SHR, compared with 6.3 microns (+/- 1.0) in WKY (P < 0.01). The central interspace between capillaries was 3.2 microns (+/- 1.6) in SHR and 6.0 microns (+/- 3.6) in WKY (P < 0.01). The venous contraction, capillary size, and capillary interspace distance correlated with the pulmonary blood pressure. The spontaneously hypertensive rat can be a model of pulmonary hypertension with its most notable structural change being increased muscularity in the small pulmonary veins.

Endothelin-1 focally constricts pulmonary veins in rats.

Serum endothelin levels increase during sepsis, ischemia, reperfusion, pulmonary operations, and systemic hypertension after surgery. Despite extensive study, the site and extent of action of endothelin on the pulmonary microcirculation are not well established. To assess the effect of endothelin on the pulmonary vasculature, especially the veins, the circulation of the lung was cast with methyl methacrylate 10 minutes after endothelin-1 was given intravenously to rats. Endothelin-1, at concentrations of 0.1, 1.0, and 10.0 micrograms/kg of body weight, increased the mean systemic arterial blood pressure 8%, 7%, and 17% (p < 0.01) and mean pulmonary arterial blood pressure 15%, 28%, and 53%, respectively (p < 0.01). The proportional increases in the pulmonary pressures were greater than those of the systemic pressures (p < 0.01). Scanning electron microscopy of cast blood vessels showed more contraction of the veins than the arteries. For doses of 0, 0.1, 1.0, and 10.0 micrograms/kg, the respective focal contraction of small veins was 6.7% (+/- 4.4), 15.4% (+/- 9.1), 23.3% (+/- 10.1), and 14.4% (+/- 9.0) of the vessel diameter (p < 0.01). In addition, the diameter of capillaries increased (p < 0.01) and the capillary interspaces decreased (p < 0.01) after endothelin administration, but not in a linear dose-dependent manner. The dose of endothelin correlated with the change in the mean systemic (r = 0.82, p < 0.01) and the mean pulmonary (r = 0.80, p < 0.01) blood pressures. The mean pulmonary pressure change correlated with the focal venous contraction on the casts (r = 0.35, p < 0.01), capillary diameter (r = 0.64, p < 0.01), and capillary interspace distance (r = -0.34, p < 0.01). The venous contraction was related to the capillary diameter (r = 0.26, p < 0.01). The most notable effect of endothelin-1 in rat pulmonary microcirculation is focal constriction of small veins. Because this effect may lead to pulmonary edema, endothelin antagonists may be of benefit in a variety of clinical situations.