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Biomedical subjects

M I Phillips

Publications and source records attributed to M I Phillips.

At least 37 records · Page 2Linked to original sources
PubMed2000

New beta-blocker: prolonged reduction in high blood pressure with beta(1) antisense oligodeoxynucleotides.

beta-Blockers are widely used for hypertension treatment but must be taken daily. We have developed a novel beta-blocker by targeting beta(1)-adrenergic receptor (beta(1)-AR) mRNA with antisense oligodeoxynucleotides (beta(1)-AS-ODN). A single intravenous injection of beta(1)-AS-ODN significantly reduced cardiac contractility and blood pressure (38+/-5 mm Hg, P<0.05) in spontaneously hypertensive rats for 3 weeks. In the present study, we improved the antihypertensive effect of beta(1)-AS-ODN by delivery with the cationic liposomes DOTAP/DOPE and studied its impact on the peripheral renin-angiotensin system. Five charge ratios (+/-) of liposome/ODN from 0 to 3.5 were tested to deliver 0. 5 mg/kg beta(1)-AS-ODN intravenously in spontaneously hypertensive rats (n=30). On the basis of the magnitude and duration of hypotension, 2.5 was determined to be the optimal charge ratio, which decreased blood pressure by up to 35 mm Hg for 20 to 33 days (P<0.05). The effects were specific for beta(1)-AR, because radioligand binding assay and quantitative autoradiography showed a 35% reduction in beta(1)-AR levels in kidney but no change in beta(2)-AR. beta(1)-AS-ODN diminished the preprorenin mRNA levels in renal cortex by 37% 4 days after administration. This transient effect was followed by a delayed yet marked diminution of plasma renin activity and plasma angiotensin II levels on days 10 and 17 (P<0.01). The results show that beta(1)-AS-ODN has an effective long-term antihypertensive effect up to 33 days with a single intravenous injection. The mechanism appears to be through reduced beta(1)-AR number specifically and reduced cardiac contractility. The inhibition of the renin-angiotensin system is probably a second mechanism to produce the sustained antihypertensive effect of beta(1)-AS-ODN.

Adrenergic beta-Antagonists↗
PubMed2000

Somatic gene therapy for hypertension.

Gene therapy for hypertension is needed for the next generation of antihypertensive drugs. Current drugs, although effective, have poor compliance, are expensive and short-lasting (hours or one day). Gene therapy offers a way to produce long-lasting antihypertensive effects (weeks, months or years). We are currently using two strategies: a) antisense oligodeoxynucleotides (AS-ODN) and b) antisense DNA delivered in viral vectors to inhibit genes associated with vasoconstrictive properties. It is not necessary to know all the genes involved in hypertension, since many years of experience with drugs show which genes need to be controlled. AS-ODN are short, single-stranded DNA that can be injected in naked form or in liposomes. AS-ODN, targeted to angiotensin type 1 receptors (AT1-R), angiotensinogen (AGT), angiotensin converting enzyme, and ss1-adrenergic receptors effectively reduce hypertension in rat models (SHR, 2K-1C) and cold-induced hypertension. A single dose is effective up to one month when delivered with liposomes. No side effects or toxic effects have been detected, and repeated injections can be given. For the vector, adeno-associated virus (AAV) is used with a construct to include a CMV promoter, antisense DNA to AGT or AT1-R and a reporter gene. Results in SHR demonstrate reduction and slowing of development of hypertension, with a single dose administration. Left ventricular hypertrophy is also reduced by AAV-AGT-AS treatment. Double transgenic mice (human renin plus human AGT) with high angiotensin II causing high blood pressure, treated with AAV-AT1-R-AS, show a normalization of blood pressure for over six months with a single injection of vector. We conclude that ODNs will probably be developed first because they can be treated like drugs for the treatment of hypertension with long-term effects. Viral vector delivery needs more engineering to be certain of its safety, but one day may be used for a very prolonged control of blood pressure.

Animals↗
PubMed2000

The potential role of antisense oligodeoxynucleotide therapy for cardiovascular disease.

Current drugs used in the treatment of cardiovascular disease are effective but compliance is poor and they are short acting (hours or one day). Gene therapy offers a way to produce long-lasting effects (weeks, months or years). Antisense inhibition is being developed for the treatment of hypertension, myocardial ischaemia and improved allograft survival in human vascular bypass grafts. We are currently using 2 strategies: (i) antisense oligodeoxynucleotides (AS-ODNs) which are delivered nonvirally and (ii) antisense DNA delivered in viral vectors to inhibit genes associated with vasoconstrictive properties. It is not necessary to know all the genes involved in hypertension, since many years of experience with drugs show which genes need to be controlled. AS-ODN are short, single-stranded DNA that can be injected in naked form or in liposomes. AS-ODN targeted to angiotensin type 1 (AT1) receptors, angiotensinogen (ATG), angiotensin converting enzyme (ACE) and beta1 adrenoceptors effectively reduce hypertension in rat models. A single dose is effective for up to one month when delivered with liposomes. No adverse or toxic effects have been detected, and repeated injections are effective. For viral delivery, adeno-associated virus (AAV) is used with a construct to include a cytomegalovirus or tissue-specific promoter, antisense DNA to ATG, ACE or AT1 receptors and a reporter gene. Results in rats and transgenic mice show significant prolonged reduction of hypertension, with a single dose administration of AAV-AS. Left ventricular hypertrophy is also reduced by antisense treatment. AS-ODNs to AT1 receptors, ATG and beta1 adrenoceptors provide cardioprotection from the effects of myocardial ischaemia. The AT1 receptor is more protective than losartan and does not increase plasma angiotensin as losartan does.

Animals↗
PubMed2000

Protection against myocardial dysfunction induced by global ischemia-reperfusion by antisense-oligodeoxynucleotides directed at beta(1)-adrenoceptor mRNA.

Plasma catecholamine levels rise, and myocardial beta(1)-adrenoceptor (beta(1)-AR) sensitivity increases during ischemia. These factors enhance myocardial injury and cardiac dysfunction. beta(1)-AR blockers are clinically used to protect heart against ischemia and to improve cardiac dysfunction in patients with ischemic heart disease, but these agents often cause intolerable side effects. To examine the potential cardioprotective effect of therapy with antisense-oligodeoxynucleotides directed at beta(1)-AR mRNA (beta(1)-AS-ODNs) during myocardial ischemia-reperfusion, Sprague-Dawley rats were treated with beta(1)-AS-ODNs or inverted-oligodeoxynucleotides (IN-ODNs), each 200 microg/rat. Hearts were excised, perfused, and subjected to global ischemia (30 min) followed by reperfusion (30 min). Other rats were given selective beta(1)-AR blocker atenolol (2 mg/kg) or saline before excising the hearts. Ischemia-reperfusion resulted in cardiac dysfunction, indicated by an increase in coronary perfusion pressure and left ventricular end-diastolic pressure and a decrease in developed left ventricular pressure, as well as evidence of lipid peroxidation in saline-treated rats (all P <.05 versus control values). Administration of AS-ODNs or atenolol, but not IN-ODNs, protected hearts against functional deterioration and lipid peroxidation (P <.05 versus saline or IN-ODNs treatment). AS-ODNs therapy appeared to be equivalent to atenolol in these effects. Expression of beta(1)-AR protein as well as mRNA in the myocardium were markedly up-regulated after ischemia-reperfusion, and treatment with beta(1)-AS-ODNs, but not atenolol, decreased the rise in enhanced expression of beta(1)-AR. These observations imply that beta(1)-AS-ODNs can ameliorate cardiac dysfunction after ischemia-reperfusion by reducing the expression of beta(1)-AR in the ischemic-reperfused myocardium.

Animals↗
PubMed2000

Designing antisense to inhibit the renin-angiotensin system.

Overactive renin-angiotensin system has been indicated in numerous pathological situations. Current treatment is based on pharmaceutical compounds, which work on the proteins level. Undisputedly helpful, it is not, however, flawless. Some of the drawbacks include adverse effects and non-compliance problem, since in many cases medicine has to be taken at least once a day for a long time. Therefore it seems logical to try a different approach, for instance to correct the disease at the gene expression level, possibly having a choice of shorter or longer-lasting effects. This current review combines results, relevant to the angiotensin system, with the antisense approach, which decreases amount of target protein by interfering at the mRNA level. Dependent on the tool used--oligodeoxynucleotide, plasmid or viral vector, the antisense effect lasts from few days to months.

Animals↗
PubMed1999

Sustained inhibition of angiotensin I-converting enzyme (ACE) expression and long-term antihypertensive action by virally mediated delivery of ACE antisense cDNA.

Angiotensin I-converting enzyme (ACE) inhibitors have been proven to be highly effective and are for the most part the drugs of choice in the treatment and control of hypertension, congestive heart failure, and left ventricular dysfunction. Despite this, questions regarding side effects and compliance with this traditional pharmacological strategy remain. In view of these observations, coupled with recent advances in gene-transfer technology, our objective in this study was to determine whether the expression of ACE could be controlled on a permanent basis at a genetic level. We argued that the introduction of ACE antisense to inhibit the enzyme would be a prerequisite in considering the antisense gene therapy for the control of hypertension and other related pathological states. Retroviral vectors (LNSV) containing ACE sense (LNSV-ACE-S) and ACE antisense (LNSV-ACE-AS) sequences were constructed and were used in rat pulmonary artery endothelial cells (RPAECs) to determine the feasibility of this approach. Infection of rat RPAECs with LNSV-ACE-S and LNSV-ACE-AS resulted in a robust expression of transcripts corresponding to ACE-S and ACE-AS, respectively, for the duration of these experiments, ie, 8 consecutive passages. The expression of ACE-AS but not of ACE-S was associated with a permanent decrease of approximately 70% to 75% in ACE expression and a 50% increase in the B(max) for the AT(1)s. Although angiotensin II caused a concentration-dependent stimulation of intracellular Ca(2+) levels in both ACE-S- and ACE-AS-expressing cells, the stimulation was significantly higher in ACE-AS-expressing RPAECs. In vivo experiments demonstrated a prolonged expression of ACE-AS transcripts in cardiovascularly relevant tissues of rats. This was associated with a long-term reduction in blood pressure by approximately 15 mm Hg, exclusively in the spontaneously hypertensive rat. These observations demonstrate that delivery of ACE-AS by retroviral vector results in a permanent inhibition of ACE and a long-term reduction in high blood pressure in the spontaneously hypertensive rat.

Angiotensin II↗
PubMed1999

Probing intracellular dynamics in living cells with near-field optics.

Near-field optics (NFO) overcomes the diffraction limit of light microscopes and permits visualization of single molecules. However, despite numerous applications of NFO in the physical sciences, there is still a paucity of applications in the neurosciences. In this work, the authors have developed NFO probes to image intracellular dynamic processes in living cells. This is the first time a NFO probe has been inserted inside a living cell to deliver light to a spatially controlled region for optical measurements and to record cellular responses to external stimuli. Two different optical detection systems (CCD camera and avalanche photon detection) were developed to monitor cellular responses to drug administration in two different cell types. NG108-15 neuroblastoma cells and vascular smooth muscle cells (VSMC) were penetrated with NFO probes. Intracellular Ca2+ increases post drug stimulation were detected by NFO probes. The cells were loaded with either fura-2/AM or fluo-3/AM calcium dyes. VSMC were stimulated with angiotensin II, resulting in a precise area of intracellular Ca2+ increase. Different response profiles of Ca2+ increases were observed after ionomycin and bradykinin administration in NG108-15 cells. Responsive heterogeneities due to ionomycin among different cells of the same type were recorded. The results show that NFO probes make possible real-time visualization of intracellular events. With refinement, intracellular NFO probes offer the potential of probing cell function with fast temporal and excellent spatial resolutions.

Angiotensin II↗
PubMed1999

The effect of ouabain on water diffusion in the rat hippocampal slice measured by high resolution NMR imaging.

High resolution NMR imaging of the isolated perfused rat hippocampal slice was used to quantitate ADC changes following ouabain-induced cell swelling. Hippocampal slices were studied in artificial cerebrospinal fluid and then in ouabain using a 600-MHz narrow bore spectrometer and a home-built perfusion chamber. The brain slices demonstrated biexponential diffusion behavior. After perfusion with 1 mMouabain, there was an increase in the fraction of slowly diffusing water. The ADCs of the two fractions did not change. These data support the hypothesis that the decrease in the ADC of brain water following an ischemic attack is caused by cell swelling. The relative amplitudes of the two diffusing fractions do not match the expected ratio of intracellular and extracellular fractions. This discrepancy may be principally due to the difference in T2 relaxation rates of the two compartments.

Animals↗
PubMed1999

MRI measurement of cell volume fraction in the perfused rat hippocampal slice.

T(1)-weighted NMR imaging of the isolated perfused rat hippo-campal slice was used to estimate cell volume fraction. Eight brain slices were studied in artificial cerebrospinal fluid (aCSF) using a 600 MHz narrow bore spectrometer and a home built perfusion chamber. Cell volume fraction was calculated as 1 - f(ECS), where f(ECS) is the distribution volume of gadodiamide in the slice. This was determined by measuring the T(1) of the slice before and after perfusion with gadodiamde. A mean cell volume fraction of 0.66 +/- 0. 04 was estimated. The addition of 60 mM mannitol to three of the brain slices produced a 26% decrease in the cell volume fraction. The technique affords a simple means of estimating cell volume fraction and can be extended to produce images reflecting cell density. Magn Reson Med 42:603-607, 1999.

Animals↗
PubMed1999

In vivo dynamics and distribution of intracerebroventricularly administered gadodiamide, visualized by magnetic resonance imaging.

Direct injections into the cerebroventricles have been extensively utilized in neurophysiological studies. Mapping the distribution of injectate after intracerebroventricular injection has been made only by post mortem analysis, and the dynamic distribution of injectate within the brain has not been well characterized. In this report, we apply contrast-enhanced magnetic resonance imaging to study the pharmacokinetics and extent of non-ionic gadodiamide transport into brain tissue in vivo after intracerebroventricular administration. The results indicate that intracerebroventricular injectate travels quickly throughout the ventricular system from the lateral ventricular site of injection to the fourth ventricle and foramina of Luschka and Magendie within 2 min. After this, the signal intensity begins to increase in the periventricular and paraventricular brain parenchyma. Contrast enhancement is visible 2 mm into the brain tissue from the ventricles. Quantitative analysis of the data shows that the transport of gadodiamide across the ependymal layer that lines the cerebrospinal fluid space characterized a rate constant of 0.066+/-0.017 min(-1). These results provide a better understanding of chemical transport and diffusion following direct injection into the cerebroventricles. They provide information on the in vivo dynamics of injectate after intracerebroventricular administration, and show that contrast enhanced magnetic resonance imaging may be used to more precisely define the target sites of chemicals after intracerebroventricular administration into the brain.

Animals↗
PubMed1999

Nuclear magnetic resonance imaging measurements of water diffusion in the perfused hippocampal slice during N-methyl-D-aspartate-induced excitotoxicity.

Significant changes in the apparent diffusion coefficient of water are observed in nuclear magnetic resonance images of patients with acute ischemic stroke. However, the underlying mechanisms of these apparent diffusion coefficient changes are still unresolved. To analyse possible mechanisms, this study applies nuclear magnetic resonance imaging on a 14.1 Tesla narrow-bore magnet to quantitatively study water diffusion in individually perfused brain slices following exposure to N-methyl-D-aspartate excitotoxicity. The results indicate that brain slices have at least two distinct diffusing water compartments with apparent diffusion coefficients of 0.96+/-0.10x10(-3) mm2/s and 0.06+/-0.01x10(-3) mm2/s. When excitotoxicity was induced with N-methyl-D-aspartate, there was a significant decrease in the fraction of the fast diffusing water component in the slices (P<0.001). However, neither apparent diffusion coefficient changed significantly. Prior treatment with dizocilpine maleate (MK-801) depressed the effects of N-methyl-D-aspartate (P<0.01, ANOVA). The results demonstrate brain slice compartmental changes resulting from direct receptor stimulation and provide evidence for tissue water redistribution as an important mechanism for changes in apparent diffusion coefficient seen in clinical magnetic resonance imaging. The brain slice preparation affords a well-controlled method to study the mechanisms of tissue nuclear magnetic resonance contrast, bridging the gap between basic nuclear magnetic resonance studies and clinical magnetic resonance imaging. The brain slice model also offers a new way to test the utility of potential anti-stroke drugs using high field nuclear magnetic resonance imaging.

Algorithms↗
PubMed1999

NMR microscopy--beginnings and new directions.

In this paper we briefly review the origins of NMR microscopy, and in the spirit of the Sir Peter Mansfield Symposium of which this presentation was a part, point out especially Sir Mansfield and his co-workers contributions in this area. We then review some recent studies applying magnetic resonance (MR) microscopy focusing on our own contributions in these regards, in particular with reference to imaging of single neurons and more recent microimaging studies on isolated perfused brain slices. Finally we briefly describe recent preliminary studies on the feasibility of spectroscopic experiments that may be performed at the single cell level, further illustrating the growing scope and potential of magnetic resonance imaging (MRI) in general as a tool for examining biological systems non-invasively.

Animals↗
PubMed1999

Intravenous angiotensinogen antisense in AAV-based vector decreases hypertension.

Angiotensinogen (AGT) has been linked to hypertension. Because there are no direct inhibitors of AGT, we have developed antisense (AS) inhibition of AGT mRNA delivered in an adeno-associated virus (AAV)-based plasmid vector. This plasmid, driven by the cytomegalovirus promoter, contains a green fluorescent protein reporter gene and AS cDNA for rat AGT. Transfection of the plasmid into rat hepatoma cells brought a strong expression of the transgenes and a significant reduction in the level of AGT. In the in vivo study, naked plasmid DNA was intravenously injected into adult spontaneously hypertensive rats at different doses (0.6, 1.5, and 3 mg/kg). Expression of AGT AS mRNA was present in liver and heart, and it lasted longer in the liver. All three doses produced a significant decrease in blood pressure (BP). BP decreased for 2, 4, and 6 days, respectively. The lowest dose decreased BP by 12 +/- 3.0 mmHg, whereas the higher doses decreased BP by up to 22.5 +/- 5.2 mmHg compared with the control rats injected with saline (P < 0.01). The injection of the plasmid with liposomes produced a more profound and longer reduction (8 days) in BP. Consistent changes in plasma AGT level were observed. Sense plasmid had no effect. No liver toxicity was observed after injection of AS plasmid with or without liposomes. Our results suggest that the systemic delivery of AS against AGT mRNA by AAV-based plasmid vector, especially with liposomes, may have potential for gene therapy of hypertension and that further studies with the plasmid packaged into a recombinant AAV vector for a longer-lasting AS effect are warranted.

Angiotensinogen↗
PubMed1999

Antisense inhibition of AT1 receptor in vascular smooth muscle cells using adeno-associated virus-based vector.

Vascular smooth muscle cells (VSMCs) are the main peripheral target for vasoconstriction and growth-promoting activity of angiotensin II (Ang II), acting through angiotensin type 1 receptors (AT1-R). Current antihypertension treatments include daily reductions in the effects of Ang II. To decrease an effect of Ang II in a prolonged fashion, we have developed an adeno-associated virus (AAV) vector with antisense DNA for AT1-R. AAV has many advantages over other viral vectors. AAV is nonpathogenic, does not stimulate inflammation or immune reaction and enters nondividing cells, and provides stable long-term gene expression. To test AAV in VSMCs, we constructed and tested plasmid AAV (pAAV) and recombinant AAV (rAAV) with AT1-R antisense DNA. rAAV was constructed with a cassette containing a cytomegalovirus promoter and the cDNA for the AT1-R inserted in the antisense direction. The cassette was packaged into the virion. Transfection of VSMCs with the pAAV antisense to AT1-R produced a significant reduction in the amount of AT1-R (P<0.01). Transduction of VSMCs with the rAAV-AT1-R-AS at MOI of 5 also showed significant reduction of AT1-R and long-lasting expression of the transgene for at least 8 weeks. The reduction of AT1-R number in VSMCs was concomitant with a decrease in the Ang II-stimulated increase of intracellular calcium. The results show that AAV vector delivers AT1-R antisense to inhibit AT1-R in VSMCs. For the purpose of gene therapy for hypertension, it is necessary to demonstrate the effectiveness of a vector system in VSMCs. This study provides support for the potential use of AAV AT1-R antisense in VSMCs.

Angiotensin II↗
PubMed1998

Angiotensin II in central nervous system physiology.

In summary, the prevailing concept is that brain Ang II increases blood pressure by activating AT1 receptors, and that these have a neuromodulating effect to increase the activity of autonomic nervous system. Pathways for Ang II stimulating thirst and blood pressure, increased vasopressin release and sympathetic activation have been outlined. Brain RAS synthesis, while incompletely understood, is active in the absence of a peripheral RAS. Angiotensin elicits specific receptor mediated signals in neurons, particularly in the hypothalamus and brainstem. These actions are due to neuronal membrane ionic currents and the regulation of transcription factors. The areas to be explored further are characterization and functional roles of the other AT receptor subtypes, such as AT4, AT(1-7) and nuclear AT-R. Their interactions with other peptides and transmitters, and their signaling pathways need to be investigated. The story that began 100 years ago with renin is certainly not ended and will continue to unfold as further investigations with new techniques progress.

Angiotensin II↗