James P. Morgan

Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia

Preeclampsia, a syndrome affecting 5% of pregnancies, causes substantial maternal and fetal morbidity and mortality. The pathophysiology of preeclampsia remains largely unknown. It has been hypothesized that placental ischemia is an early event, leading to placental production of a soluble factor or factors that cause maternal endothelial dysfunction, resulting in the clinical findings of hypertension, proteinuria, and edema. Here, we confirm that placental soluble fms-like tyrosine kinase 1 (sFlt1), an antagonist of VEGF and placental growth factor (PlGF), is upregulated in preeclampsia, leading to increased systemic levels of sFlt1 that fall after delivery. We demonstrate that increased circulating sFlt1 in patients with preeclampsia is associated with decreased circulating levels of free VEGF and PlGF, resulting in endothelial dysfunction in vitro that can be rescued by exogenous VEGF and PlGF. Additionally, VEGF and PlGF cause microvascular relaxation of rat renal arterioles in vitro that is blocked by sFlt1. Finally, administration of sFlt1 to pregnant rats induces hypertension, proteinuria, and glomerular endotheliosis, the classic lesion of preeclampsia. These observations suggest that excess circulating sFlt1 contributes to the pathogenesis of preeclampsia.

Differential Cardiac Effects of Growth Hormone and Insulin-like Growth Factor1 in the Rat A Combined In Vivo and In Vitro Evaluation

BACKGROUND Despite their increasing clinical use and recent evidence that growth hormone (GH) and insulin-like growth factor-1 (IGF-1) target the heart, there has been no systematic investigation of the effects of GH and IGF-1 on the cardiovascular system. METHODS AND RESULTS Sixty normal but growing adult female rats were randomized to receive 4 weeks of treatment with GH (3.5 mg.kg-1.d-1), IGF-1 (3 mg.kg-1.d-1), a combination of the two, or placebo. Transthoracic echocardiograms were performed at baseline and at 2 weeks and 4 weeks of treatment. After the final echocardiography, rats underwent either closed-chest left ventricular (LV) catheterization or Langendorff perfusion studies. Myocyte diameter and interstitial tissue fraction were assessed by morphometric histology. Echocardiographic and ex vivo data demonstrated a LV hypertrophic response in all three groups of treated animals that was most marked in the GH group, which alone exhibited a concentric growth pattern (relative wall thickness, 0.52 versus 0.42 to 0.44 in the other groups; P < .001). At 4 weeks, cardiac index was significantly higher and total systemic vascular resistance was lower in all groups of treated animals than in control animals (both P < .001), whereas arterial blood pressure did not differ significantly. All indexes of in vivo and in vitro cardiac function were higher in GH- and IGF-1-treated rats than in control animals, whereas combination therapy yielded a blunted effect. Myocyte diameter was increased in all three treated groups without an increase in interstitial tissue. CONCLUSIONS Exogenous administration of GH and IGF-1 in the normal adult rat induces a cardiac hypertrophic response without development of significant fibrosis. Cardiac performance is increased both in vivo and in the isolated heart.

Deletion of Mineralocorticoid Receptors From Macrophages Protects Against Deoxycorticosterone/Salt-Induced Cardiac Fibrosis and Increased Blood Pressure

Increased mineralocorticoid levels plus high salt promote vascular inflammation and cardiac tissue remodeling. Mineralocorticoid receptors are expressed in many cell types of the cardiovascular system, including monocytes/macrophages and other inflammatory cell types. Although mineralocorticoid receptors are expressed in monocytes/macrophages, their role in regulating macrophage function to date has not been investigated. We, thus, used the Cre/LoxP-recombination system to selectively delete mineralocorticoid receptors from monocytes/macrophages with the lysozyme M promoter used to drive Cre expression (MRflox/flox/LysMCre/− mice). Male mice from each genotype (MRflox/flox or wild-type and MRflox/flox/LysMCre/− mice) were uninephrectomized, given 0.9% NaCl solution to drink, and treated for 8 days or 8 weeks with either vehicle (n=10) or deoxycorticosterone (n=10). Equivalent tissue macrophage numbers were seen for deoxycorticosterone treatment of each genotype at 8 days; in contrast, plasminogen activator inhibitor type 1 and NAD(P)H oxidase subunit 2 levels were increased in wild-type but not in MRflox/flox/LysMCre/− mice given deoxycorticosterone. Baseline expression of other inflammatory genes was reduced in MRflox/flox/LysMCre/− mice compared with wild-type mice. At 8 weeks, deoxycorticosterone-induced macrophage recruitment and connective tissue growth factor and plasminogen activator inhibitor type 1 mRNA levels were similar for each genotype; in contrast, MRflox/flox/LysMCre/− mice showed no increase in cardiac fibrosis or blood pressure, as was seen in wild-type mice at 8 weeks. These data demonstrate the following points: (1) mineralocorticoid receptor signaling regulates basal monocyte/macrophage function; (2) macrophage recruitment is not altered by loss of mineralocorticoid receptor signaling in these cells; and (3) a novel and significant role is seen for macrophage signaling in the regulation of cardiac remodeling and systolic blood pressure in the deoxycorticosterone/salt model.

Growth Hormone Attenuates Early Left Ventricular Remodeling and Improves Cardiac Function in Rats With Large Myocardial Infarction

OBJECTIVES We sought to investigate the cardiac effects of growth hormone (GH) administration during the early phase of pathologic remodeling in a rat model of large myocardial infarction (MI). BACKGROUND Recent evidence suggests that exogenous administration of GH evokes a hypertrophic response and increases left ventricular (LV) function in vivo in rats with normal or chronically failing hearts. We hypothesized that these effects would attenuate ventricular remodeling early after MI. METHODS Fifty-eight male rats underwent sham operation (n = 19) or had induced MI (n = 39). The day after the operation, the infarcted rats were randomized to receive 3 weeks of treatment with GH, 3 mg/kg body weight per day (n = 19) or placebo (n = 20). Echocardiography, catheterization and isolated whole heart preparations were used to define cardiac structure and function. RESULTS Growth hormone caused hypertrophy of the noninfarcted myocardium in a concentric pattern, as noted by higher echocardiographic relative wall thickness at 3 weeks and by morphometric histologic examination. Left ventricular dilation was reduced in the GH-treated versus placebo group (echocardiographic LV diastolic diameter to body weight ratio 2.9 +/- 0.1 vs. 3.5 +/- 0.2 cm/kg; p < 0.05). In vivo and in vitro cardiac function was improved after GH treatment. Despite elevated insulin-like growth factor-1 (IGF-1) serum levels in GH-treated rats, myocardial IGF-I messenger ribonucleic acid was not different among the three groups, suggesting that an increase in its local expression does not appear necessary to yield the observed effects. CONCLUSIONS These data demonstrate that early treatment of large MI with GH attenuates the early pathologic LV remodeling and improves LV function.

Significant improvement of heart function by cotransplantation of human mesenchymal stem cells and fetal cardiomyocytes in postinfarcted pigs

BACKGROUND Viable cardiomyocytes after myocardial infarction (MI) are unable to repair the necrotic myocardium due to their limited capability of regeneration. The present study investigated whether intramyocardial transplantation of human mesenchymal stem cells (hMSCs) or cotransplantation of hMSCs plus human fetal cardiomyocytes (hFCs; 1:1) reconstituted impaired myocardium and improved cardiac function in MI pigs. METHODS AND RESULTS Cultured hMSCs were transfected with green fluorescent protein (GFP). Six weeks after MI induction and cell transplantation, cardiac function was significantly improved in MI pigs transplanted with hMSCs alone. However, the improvement was even markedly greater in MI pigs cotransplanted with hMSCs plus hFCs. Histological examination demonstrated that transplantation of hMSCs alone or hMSCs plus hFCs formed GFP-positive engrafts in infarcted myocardium. In addition, immunostaining for cardiac alpha-myosin heavy chain and troponin I showed positive stains in infarcted regions transplanted with hMSCs alone or hMSCs plus hFCs. CONCLUSIONS Our data demonstrate that transplantation of hMSCs alone improved cardiac function in MI pigs with a markedly greater improvement from cotransplantation of hMSCs plus hFCs. This improvement might result from myocardial regeneration and angiogenesis in injured hearts by engrafted cells.

Abnormal cardiac function in the streptozotocin-induced, non–insulin-dependent diabetic rat: Noninvasive assessment with Doppler echocardiography and contribution of the nitric oxide pathway☆

OBJECTIVES We sought to evaluate in vivo and in vitro left ventricular (LV) geometry and function in streptozotocin-induced diabetic rats and the possible role of the nitric oxide (NO) pathway. BACKGROUND Diabetes results in cardiac dysfunction; however, the specific abnormalities are unknown. Because decreased NO contributes to abnormal vascular function in diabetics, we hypothesized that NO pathway abnormalities may contribute to diabetic cardiomyopathy. METHODS Control rats and those with non-insulin-dependent diabetes mellitus (NIDDM) underwent echocardiography, hemodynamic assessment, isolated heart perfusion and measurement of exhaled NO and LV endothelial constitutive nitric oxide synthase (ecNOS). RESULTS Diabetic rats had increased LV mass (3.3 +/- 0.6 vs. 2.6 +/- 0.3 g/g body weight [BW], p < 0.001) and cavity dimensions (diastolic 2.0 +/- 0.1 vs. 1.8 +/- 0.2 cm/cm tibial length [TL], p < 0.05). Diabetic rats had prolonged isovolumic relaxation time (IVRT) (40 +/- 8 vs. 26 +/- 6 ms, p < 0.0001), increased atrial contribution to diastolic filling (0.47 +/- 0.09 vs. 0.30 +/- 0.08 m/s, p < 0.0001), and elevated in vivo LV end-diastolic pressure (7 +/- 6 vs. 2 +/- 1 mm Hg, p = 0.04). Diabetic rats had increased chamber stiffness. Shortening was similar in both groups, despite reduced meridional wall stress in diabetics, suggesting impaired systolic contractility. Exhaled NO was lower in diabetic rats (1.8 +/- 0.2 vs. 3.3 +/- 0.3 parts per billion, p < 0.01) and correlated with Doppler LV filling. The ecNOS was similar between the groups. CONCLUSIONS Diabetic cardiomyopathy is characterized by LV systolic and diastolic dysfunction, the latter correlating with decreased exhaled NO. The NO pathway is intact, suggesting impaired availability of NO as contributor to cardiomyopathy.

Calcium and cardiovascular function: Intracellular calcium levels during contraction and relaxation ofmammalian cardiac and vascular smooth muscle as detected with aequorin

Calcium ion (Ca++) plays a central role in excitation-contraction coupling of both cardiac and vascular smooth muscles and have important functional interactions with other cations, including sodium, potassium, and magnesium. Ca++ transients associated with contraction-relaxation cycles of the heart and vasculature can now be recorded directly by use of aequorin, a bioluminescent protein that emits light when it combines with Ca++. After microinjection or chemical loading of aequorin into the sarcoplasm, light output provides an index of intracellular [Ca++]. In cardiac muscle, intracellular Ca++ increases more quickly than tension and decreases toward basal levels by the time peak tension is reached. The calcium transients of working myocardium in both human subjects and other mammalian species appear to be dominated by the release and uptake of Ca++ from intracellular stores under all conditions studied. Drugs and disease states produce marked changes in the amplitude and time course of the Ca++ transient and the corresponding contractile response. In vascular smooth muscle, there are stimulus-specific patterns in intracellular Ca++ associated with tonic contractions. Although Ca++ is related to tension development, the relationship appears to be more complex than that in cardiac muscle. As a result, tension development cannot be used as an index of free Ca++ levels in vascular smooth muscle. Selection of the most effective therapy to reverse a tonic contraction in states of spasm or hypertension may depend on the specific stimulus that caused the increased tone.

Gait dynamics in mouse models of Parkinson's disease and Huntington's disease

BackgroundGait is impaired in patients with Parkinsons disease (PD) and Huntingtons disease (HD), but gait dynamics in mouse models of PD and HD have not been described. Here we quantified temporal and spatial indices of gait dynamics in a mouse model of PD and a mouse model of HD.MethodsGait indices were obtained in C57BL/6J mice treated with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 30 mg/kg/day for 3 days) for PD, the mitochondrial toxin 3-nitropropionic acid (3NP, 75 mg/kg cumulative dose) for HD, or saline. We applied ventral plane videography to generate digital paw prints from which indices of gait and gait variability were determined. Mice walked on a transparent treadmill belt at a speed of 34 cm/s after treatments.ResultsStride length was significantly shorter in MPTP-treated mice (6.6 ± 0.1 cm vs. 7.1 ± 0.1 cm, P < 0.05) and stride frequency was significantly increased (5.4 ± 0.1 Hz vs. 5.0 ± 0.1 Hz, P < 0.05) after 3 administrations of MPTP, compared to saline-treated mice. The inability of some mice treated with 3NP to exhibit coordinated gait was due to hind limb failure while forelimb gait dynamics remained intact. Stride-to-stride variability was significantly increased in MPTP-treated and 3NP-treated mice compared to saline-treated mice. To determine if gait disturbances due to MPTP and 3NP, drugs affecting the basal ganglia, were comparable to gait disturbances associated with motor neuron diseases, we also studied gait dynamics in a mouse model of amyotrophic lateral sclerosis (ALS). Gait variability was not increased in the SOD1 G93A transgenic model of ALS compared to wild-type control mice.ConclusionThe distinct characteristics of gait and gait variability in the MPTP model of Parkinsons disease and the 3NP model of Huntingtons disease may reflect impairment of specific neural pathways involved.

In vivo MRI of embryonic stem cells in a mouse model of myocardial infarction

The therapeutic potential of administering stem cells to promote angiogenesis and myocardial tissue regeneration after infarction has recently been demonstrated. Given the advantages of using embryonic stem cells and mouse models of myocardial infarction for furthering the development of this therapeutic approach, the purpose of this study was to determine if embryonic stem cells could be loaded with superparamagnetic iron oxide (SPIO) particles and imaged in a mouse model of myocardial infarction over time using MRI. Mouse embryonic stem cells were labeled with SPIO particles. When incubated with 11.2, 22.4, and 44.8 μg Fe/ml of SPIO particles, cells took up increasing amounts of iron oxide. Embryonic stem cells loaded with SPIO compared to unlabeled cells had similar viability and proliferation profiles for up to 14 days. Free SPIO injected into infarcted myocardium was not observable within 12 hr after injection. After injection of three 10‐μl aliquots of 107 SPIO‐loaded cells/ml into infarcted myocardium, MRI demonstrated that the mouse embryonic stem cells were observable and could be seen for at least 5 weeks after injection. These findings support the ability of MRI to test the long‐term therapeutic potential of embryonic stem cells in small animals in the setting of myocardial infarction. Magn Reson Med 52:1214–1219, 2004.

Cytochrome P450: a novel system modulating Ca2+ channels and contraction in mammalian heart cells

1 Cytochrome P450 (P450) is a ubiquitous enzyme system that catalyses oxidative reactions of numerous endogenous and exogenous compounds. The modulatory effects of P450 on the L‐type Ca2+ current (ICa), intracellular free Ca2+ signals and cell shortening were assessed in adult rat single ventricular myocytes. 2 Bath administration of the imidazole antimycotics, clotrimazole, econazole and miconazole, which are potent P450 inhibitors, significantly suppressed cardiac ICa. While the Ca2+ channel antagonist nifedipine blocked ICa within 30 s, clotrimazole‐induced suppression of ICa required 5.1 ± 0.4 min (n= 14) to reach a steady low level. The suppression of ICa was dose dependent and recovered after washout of clotrimazole. Intracellular dialysis with the P450 antibody anti‐rat CYP1A2 also significantly reduced cardiac ICa. 3 Additional administration of the β‐adrenergic agonist isoprenaline (1 μM) or the membrane‐permeable 8‐bromo‐cAMP (2 mM) completely reversed the suppressant effects of clotrimazole and NaCN on ICa. In addition, intracellular dialysis with 2 mM cAMP abolished the P450 inhibitor‐induced suppression of ICa. Phosphorylation of the channel with hydrolysis‐resistant ATPγS prevented the suppressant effect of clotrimazole on ICa. Furthermore, dephosphorylation of the Ca2+ channel with intracellular dialysis with phosphatase types I and II reduced ICa by 85 ± 3 % and abolished clotrimazole‐induced suppression of ICa. 4 Extracellular administration of the phospholipase A2 inhibitors mepacrine and 4‐bromophenacyl bromide significantly suppressed ICa. 5 Clotrimazole, econazole, miconazole and CN− also significantly inhibited intracellular free Ca2+ signals and cell shortening in rat single ventricular myocytes. 6 Intracellular cAMP content was significantly reduced in isolated ventricular myocytes incubated with clotrimazole or CN−. Extracellular administration of 11, 12‐epoxyeicosatrienoic acid, one of the P450‐mediated metabolites of arachidonic acid, enhanced ICa and intracellular cAMP content. The epoxyeicosatrienoic acid also restored the amplitude of the reduced ICa in P450 antibody‐dialysed myocytes. 7 The present data suggest that cytochrome P450 modulates cardiac ICa and cell contraction, and the modulation may result from changes in intracellular levels of cAMP by P450‐ mediated metabolites of arachidonic acid.