Prolactin alters blood pressure by modulating the activity of endothelial nitric oxide synthase.

Increased levels of a cleaved form of prolactin (molecular weight 16 kDa) have been associated with preeclampsia. To study the effects of prolactin on blood pressure (BP), we generated male mice with a single-copy transgene (Tg; inserted into the hypoxanthine-guanine phosphoribosyltransferase locus) that enables inducible hepatic production of prolactin and its cleavage product. The Tg is driven by the indole-3-carbinol (I3C)-inducible rat cytochrome P450 1A1 promoter. When the Tg mice were fed normal chow (NC), plasma prolactin concentrations were comparable to those in female WT mice in the last third of pregnancy, and BP was lower than in WT mice (∼95 mm Hg vs. ∼105 mm Hg). When the Tg mice were fed chow containing IC3, plasma prolactin concentrations increased threefold, BP increased to ∼130 mm Hg, and cardiac function became markedly impaired. IC3 chow did not affect the WT mice. Urinary excretion of nitrite/nitrate and the amount of Ser1177-phosphorylated endothelial nitric oxide (NO) synthase (eNOS) were significantly greater in the Tg mice fed NC than in WT mice, as they are during pregnancy. However, when I3C was fed, these indicators of NO production became significantly less in the Tg mice than in WT mice. The effects of increased plasma prolactin were abolished by a genetic absence of eNOS. Thus, a threefold increase in plasma prolactin is sufficient to increase BP significantly and to markedly impair cardiac function, with effects mediated by NO produced by eNOS. We suggest that pregnant women with abnormally high prolactin levels may need special attention.

High Elmo1 expression aggravates and low Elmo1 expression prevents diabetic nephropathy.

Human genome-wide association studies have demonstrated that polymorphisms in the engulfment and cell motility protein 1 gene (ELMO1) are strongly associated with susceptibility to diabetic nephropathy. However, proof of causation is lacking. To test whether modest changes in its expression alter the severity of the renal phenotype in diabetic mice, we have generated mice that are type 1 diabetic because they have the Ins2(Akita) gene, and also have genetically graded expression of Elmo1 in all tissues ranging in five steps from ∼30% to ∼200% normal. We here show that the Elmo1 hypermorphs have albuminuria, glomerulosclerosis, and changes in the ultrastructure of the glomerular basement membrane that increase in severity in parallel with the expression of Elmo 1. Progressive changes in renal mRNA expression of transforming growth factor ß1 (TGFß1), endothelin-1, and NAD(P)H oxidase 4 also occur in parallel with Elmo1, as do the plasma levels of cystatin C, lipid peroxides, and TGFß1, and erythrocyte levels of reduced glutathione. In contrast, Akita type 1 diabetic mice with below-normal Elmo1 expression have reduced expression of these various factors and less severe diabetic complications. Remarkably, the reduced Elmo1 expression in the 30% hypomorphs almost abolishes the pathological features of diabetic nephropathy, although it does not affect the hyperglycemia caused by the Akita mutation. Thus, ELMO1 plays an important role in the development of type 1 diabetic nephropathy, and its inhibition could be a promising option for slowing or preventing progression of the condition to end-stage renal disease.

Transforming growth factor-ß1 and diabetic nephropathy.

Transforming growth factor-ß1 (TGF-ß1) is established to be involved in the pathogenesis of diabetic nephropathy. The diabetic milieu enhances oxidative stress and induces the expression of TGF-ß1. TGF-ß1 promotes cell hypertrophy and extracellular matrix accumulation in the mesangium, which decreases glomerular filtration rate and leads to chronic renal failure. Recently, TGF-ß1 has been demonstrated to regulate urinary albumin excretion by both increasing glomerular permeability and decreasing reabsorption in the proximal tubules. TGF-ß1 also increases urinary excretion of water, electrolytes and glucose by suppressing tubular reabsorption in both normal and diabetic conditions. Although TGF-ß1 exerts hypertrophic and fibrogenic effects in diabetic nephropathy, whether suppression of the function of TGF-ß1 can be an option to prevent or treat the complication is still controversial. This is partly because adrenal production of mineralocorticoids could be augmented by the suppression of TGF-ß1. However, differentiating the molecular mechanisms for glomerulosclerosis from those for the suppression of the effects of mineralocorticoids by TGF-ß1 may assist in developing novel therapeutic strategies for diabetic nephropathy. In this review, we discuss recent findings on the role of TGF-ß1 in diabetic nephropathy.

Low TGFß1 expression prevents and high expression exacerbates diabetic nephropathy in mice.

Nephropathy develops in many but not all patients with long-standing type 1 diabetes. Substantial efforts to identify genotypic differences explaining this differential susceptibility have been made, with limited success. Here, we show that the expression of the transforming growth factor ß1 gene (Tgfb1) affects the development of diabetic nephropathy in mice. To do this we genetically varied Tgfb1 expression in five steps, 10%, 60%, 100%, 150%, and 300% of normal, in mice with type 1 diabetes caused by the Akita mutation in the insulin gene (Ins2(Akita)). Although plasma glucose levels were not affected by Tgfb1 genotype, many features of diabetic nephropathy (mesangial expansion, elevated plasma creatinine and urea, decreased creatinine clearance and albuminuria) were progressively ameliorated as Tgfb1 expression decreased and were progressively exacerbated when expression was increased. The diabetic 10% hypomorphs had comparable creatinine clearance and albumin excretion to wild-type mice and no harmful changes in renal morphology. The diabetic 300% hypermorphs had ∼1/3 the creatinine clearance of wild-type mice, >20× their albumin excretion, ∼3× thicker glomerular basement membranes and severe podocyte effacement, matching human diabetic nephropathy. Switching Tgfb1 expression from low to high in the tubules of the hypomorphs increased their albumin excretion more than 10-fold but creatinine clearance remained high. Switching Tgfb1 expression from low to high in the podocytes markedly decreased creatinine clearance, but minimally increased albumin excretion. Decreasing expression of Tgfb1 could be a promising option for preventing loss of renal function in diabetes.

Endothelin-1 critically influences cardiac function via superoxide-MMP9 cascade.

We have generated low-expressing and high-expressing endothelin-1 genes (L and H) and have bred mice with four levels of expression: L/L, ∼20%; L/+, ∼65%; +/+ (wild type), 100%; and H/+, ∼350%. The hypomorphic L allele can be spatiotemporally switched to the hypermorphic H allele by Cre-loxP recombination. Young adult L/L and L/+ mice have dilated cardiomyopathy, hypertension, and increased plasma volumes, together with increased ventricular superoxide levels, increased matrix metalloproteinase 9 (Mmp9) expression, and reduced ventricular stiffness. H/+ mice have decreased plasma volumes and significantly heavy stiff hearts. Global or cardiomyocyte-specific switching expression from L to H normalized the abnormalities already present in young adult L/L mice. An epithelial sodium channel antagonist normalized plasma volume and blood pressure, but only partially corrected the cardiomyopathy. A superoxide dismutase mimetic made superoxide levels subnormal, reduced Mmp9 overexpression, and substantially improved cardiac function. Genetic absence of Mmp9 also improved cardiac function, but increased superoxide remained. We conclude that endothelin-1 is critical for maintaining normal contractile function, for controlling superoxide and Mmp9 levels, and for ensuring that the myocardium has sufficient collagen to prevent overstretching. Even a modest (∼35%) decrease in endothelin-1 gene (Edn1) expression is sufficient to cause cardiac dysfunction.

Transforming growth factor beta1 and aldosterone.

PURPOSE OF REVIEW: It is well established that blocking the renin-angiotensin-aldosterone system (RAAS) is effective for the treatment of cardiovascular and renal complications in hypertension and diabetes mellitus. Although the induction of transforming growth factor beta1 (TGFbeta1) by components of the RAAS mediates the hypertrophic and fibrogenic changes in cardiovascular-renal complications, it is still controversial as to whether TGFbeta1 can be a target to prevent such complications. Here, we review recent findings on the role of TGFbeta1 in fluid homeostasis, focusing on the relationship with aldosterone. RECENT FINDINGS: TGFbeta1 suppresses the adrenal production of aldosterone and renal tubular sodium reabsorption. We have generated mice with TGFbeta1 mRNA expression graded in five steps, from 10 to 300% of normal, and found that blood pressure and plasma volume are negatively regulated by TGFbeta1. Notably, the 10% hypomorph exhibits primary aldosteronism and sodium and water retention due to markedly impaired urinary excretion of water and electrolytes. SUMMARY: These results identify TGFbeta signalling as an important counterregulatory system against aldosterone. Understanding the molecular mechanisms for the suppressive effects of TGFbeta1 on adrenocortical and renal function may further our understanding of primary aldosteronism, as well as assist in the development of novel therapeutic strategies for hypertension.

Harvesting, identification and barrier function of human lung microvascular endothelial cells.

Endothelial barrier dysfunction is an important contributor to the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Even though approaches that target the prevention and repair of endothelial barrier dysfunction are clearly needed, our understanding of the molecular regulation of pulmonary microvascular endothelial permeability remains incomplete. Cultured pulmonary microvascular endothelial cells represent an attractive paradigm for the study of barrier function. Here, we describe a method for the harvest, identification and culture of human lung microvascular endothelial cells (HLMVEC). HLMVEC thus obtained, grow as a monolayer, exhibit contact inhibition and have the typical cobblestone appearance. They express endothelial proteins, such as von Willebrand factor and endothelial nitric oxide synthase and take up an acetylated LDL. Furthermore, HLMVEC respond predictably and with superior sensitivity to the barrier disruptive effects of Gram positive and Gram negative bacterial products, thrombin, vascular endothelial growth factor and microtubule disrupting agents. These HLMVEC present an in-house-derived alternative to commercially available human cells for the study of mechanisms contributing to ALI and ARDS.

Fungal colonization of a pneumonectomy stump.

A 59-year-old woman developed mild recurring hemoptysis once a week for several months after a fall with trauma to the chest. Sixteen years earlier she had undergone a right pneumonectomy at a hospital elsewhere for sequelae of pulmonary tuberculosis. Bronchoscopy, performed because of the recent hemoptysis, showed material in the pneumonectomy stump. The material had a gelatinous appearance, green color with a pale margin, and oblique striations. The material was removed by grasping with forceps and withdrawing the bronchoscope. Grocott methanamine silver stain was positive for septate, nonpigmented fungal organisms. Anatomic pathology microscopy also showed mucous, acute inflammatory cells, and necrotic tissue. Cytopathology of washings from the bronchial stump showed rare degenerated benign bronchial epithelial cells and fungal hyphae. Acid fast bacilli smears and cultures were negative. Bacterial cultures showed 3+ Pseudomonas aeruginosa. The patient had no further hemoptysis.

Cardiac surgery after mediastinal radiation: extent of exposure influences outcome.

OBJECTIVES: Mediastinal radiation for thoracic malignancies uses multiple treatment fields and doses. We investigated whether more extensive radiation exposure is associated with more hospital complications and worse survival after cardiac surgery. METHODS: From January 2000 to January 2005, 230 patients underwent cardiac surgery after 3 levels of mediastinal radiation: extensive (Hodgkin disease, thymoma, and testicular cancer; n = 70), variable (eg, non-Hodgkin lymphoma and lung cancer; n = 35); and tangential (breast cancer; n = 125). Hospital complications were recorded prospectively, and time-related survival was assessed by patient follow-up (mean follow-up, 2.2 +/- 1.4 years). RESULTS: Patients receiving extensive exposure were youngest (51 vs 64 vs 72 years), with the longest radiation-to-operation interval (25 vs 13 vs 14 years), and had the most diastolic dysfunction, left main stenosis of greater than 70% (21% vs 9% vs 8%), and aortic regurgitation (79% vs 54% vs 50%). Patients receiving extensive and variable exposure had the poorest pulmonary function (percent predicted forced expiratory volume in 1 second, 57% vs 54% vs 67%; percent predicted forced vital capacity, 56% vs 63% vs 66%). All groups received a similar mix of cardiac procedures. Hospital deaths (13% vs 8.6% vs 2.4%) and respiratory complications (24% vs 20% vs 9.6%) were higher after more extensive radiation, and survival was poorer (4-year survival, 64% vs 57% vs 80%) than for patients receiving tangential radiation exposure, and it deviated more from expected matched-population life tables. CONCLUSIONS: Among patients undergoing cardiac surgery after thoracic radiation, radiation exposure is heterogeneous, and therefore these patients cannot be managed and assessed as a single uniform cohort. Extensively irradiated patients are more likely to develop radiation heart disease, which increases perioperative morbidity and decreases short- and long-term survival.

The role of 111indium-octreotide brain scintigraphy in the diagnosis of cranial, dural-based meningiomas.

OBJECTIVE: Meningiomas are common brain tumors with somatostatin receptors that bind octreotide. We report the use of (111)indium-octreotide brain scintigraphy (OBS) for the non-invasive differentiation of meningiomas from other cranial dural-based pathology. METHODS: A retrospective analysis of our experience with OBS for non-invasive identification of meningiomas was performed. Two neuroradiologists, blinded to clinical data, utilized a standardized grading scheme to define the uptake of octreotide at 6 and 24 h post-administration. The correlation between (18) F-fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET), magnetic resonance imaging (MRI) scans, and octreotide uptake was assessed. RESULTS: The cohort consisted of 50 patients having a mean age of 62.4 years and a median follow-up time of 24 months. Management consisted of biopsy (n = 4); resection (n = 10); observation (n = 16); radiosurgery (n = 21); and external beam radiotherapy (n = 3). OBS was correlated with MRI (n = 50); FDG-PET brain studies (n = 38); histology (n = 14), and angiography (n = 1). In cases where definitive diagnosis could be made, the sensitivity, specificity, positive and negative predictor values for OBS alone were 100; 50; 75; and 100, respectively. OBS provided false positive data in 3 patients (metastasis, chronic inflammation, lymphoma). Use of OBS with MRI to differentiate meningiomas from other lesions was highly significant (P < 0.001). FDG-PET correctly identified malignant pathology with 100% sensitivity and specificity. CONCLUSION: OBS may increase the diagnostic specificity of conventional MRI when differentiating meningioma from other dural-based pathologies, while the addition of FDG-PET differentiates benign from malignant lesions.

Regional referral system for patients with acute mechanical support: experience at the Cleveland Clinic Foundation.

Regional referral networks ("hub and spoke") have been created to facilitate the transfer of patients on mechanical circulatory support. Although individual centers report good success, overall outcomes have remained poor. We investigated whether preoperative variables influenced survival and could be used to help select patients best served by referral. A retrospective chart review was conducted on all patients transferred to our institution supported on cardiac assist devices. Between January 1995 and September 2003, 39 patients were received in transfer for continued care after the implantation of a cardiac assist device. Eighty-five percent of patients had the ABIOMED BVS 5000 implanted. The most common indication was postcardiotomy shock. Sixty-four percent of patients were not candidates for heart transplantation due to medical or social contraindications. The 30-day mortality of this group was 62%. Survivors had less comorbidity and were less likely to have complex surgeries, neurologic impairment, and multisystem organ failure when presenting to our center. Devices were weaned in 30% of cases. Only six patients (15%) were successfully transplanted, and five of these patients have done well at follow-up. Based on our experience, we believe that cardiogenic shock patients benefit from a regional referral system if they have not had complex cardiac surgical procedures or developed multisystem organ failure. Furthermore, there is a survival advantage when using long-term devices because this allows possible recovery or transplantation.

Ventricular assist devices and aggressive immunosuppression: looking beyond overall survival.

BACKGROUND: Patients bridged to heart transplantation with a ventricular assist device (VAD) developed coronary vasculopathy at the same rate as non-bridged patients despite having higher levels of pre-formed antibodies. We hypothesized that allosensitized VAD patients have higher levels of immunosuppression and thus different morbidity and causes of mortality. METHODS: Patients who received a transplant between January 1996 and May 2002 were separated into 2 groups based on the need for VAD support as a bridge to transplantation. Transplant and Inpatient Pharmacy Databases and charts were queried for date of transplantation, degree of allosensitization, use of desensitization therapy, immunosuppressive strategies, number of treated rejection episodes, and specific causes of death. RESULTS: This study investigated 238 patients (125 VAD patients, 113 non-VAD patients). VAD patients were more likely to be allosensitized than non-VAD patients (20% vs 5%, p < 0.01). OKT3 was given to 22% of VAD patients as anti-rejection prophylaxis and 14% received pre-transplant plasmapheresis. Non-VAD patients rarely were desensitized (2.6% of non-VAD patients). After transplantation, 68 VAD patients (54%) and 44 non-VAD patients (39%) had episodes of severe rejection requiring therapy. Episodes of rejection in VAD patients were commonly treated with steroids (90%), plasmapheresis (10%), and OKT3 (7%), and episodes of rejection in non-VAD patients were treated with steroids (76%) and OKT3 (8%). The 5-year survival for both groups was similar (90% and 86% respectively, p = 0.31). VAD patients commonly died of sepsis (75%), and non-VAD patients died of rejection (39%) and ischemic transplant cardiomyopathy (30%). CONCLUSION: When short-term outcomes between bridged and non-bridged heart transplant recipients were compared, overall survival was similar but causes of death differed. Findings in this study might aid in the post-operative management of patients bridged to transplantation with a VAD.

Early and midterm risk of coronary allograft vasculopathy in patients bridged to orthotopic heart transplantation with ventricular assist devices.

BACKGROUND: Coronary transplant vasculopathy (CAV) has been associated with both immunologic and nonimmunologic factors. The impact of preoperative ventricular assist device (VAD) support on the development of CAV has not been studied. To examine this, we obtained posttransplant coronary angiograms from a group of patients bridged with VAD and compared them to post transplant coronary angiograms of a non-VAD cohort. METHODS: Adult patients undergoing orthotopic heart transplant between 1996-2000 were retrospectively studied and divided into VAD and non-VAD patients. Coronary angiograms were retrospectively reviewed and severity of coronary vasculopathy was categorized as trivial, mild, moderate, or severe. Other variables studied included recipient and donor demographics, cytotoxic panel reactive antibodies (PRA) against T-cell targets and flow cytometric crossmatching against donor T lymphocytes. RESULTS: There was no significant difference between groups regarding demographics. However, VAD patients had a sixfold greater chance of having a T-cell PRA >10% at the time of transplant (p < 0.05), and a fourfold greater chance of having a positive cross match when compared to non-VAD patients (p < 0.05). There was no significant difference in the degree of CAV between groups. Normal coronary anatomy was present in 76% of VAD patients and 64% of non-VAD patients (p = 0.37). These results were similar at 2- and 3-year follow-up (76 vs. 74% and 80 vs. 62%, respectively). CONCLUSION: Preoperative VAD use is associated with increased sensitization; however, these patients develop CAV at the same rate as those not bridged with a VAD.