Ultrasound enhancement of cationic lipid-mediated gene transfer to primary tumors following systemic administration.

The impact of a localized application of ultrasound on gene transfer to primary tumors following systemic administration of cationic lipid based transfection complexes was investigated. We have previously shown that systemic administration of DOTMA (N-[(1-(2-3-dioleyloxy) propyl)]-N-N-N-trimethylammonium chloride):cholesterol-based transfection complexes to tumor-bearing mice resulted in expression in the tumor and other tissues, primarily the lungs. Application of ultrasound to the tumor before or after the injection resulted in a significant increase in gene transfer to the tumor with no increase observed in other tissues. The magnitude of increased expression ranged from three- to 270-fold depending upon the DNA dose. The following parameters were optimized for maximal increase: duration of ultrasound application, the time interval between plasmid injection and sonoporation, and plasmid dose. A combination of plasmid quantitation and fluorescence microscopy showed that ultrasound increased tumor uptake of the plasmid and that uptake was limited to the tumor vasculature. Using an IL- 12 expression plasmid, the combination of a single plasmid dose (10 microg) and ultrasound treatment produced significantly higher levels of IL-12 in tumor. This increased expression was sufficient to inhibit tumor growth compared with the control conditions. These data demonstrate the potential application of sonoporation as an effective method for enhancing the expression of systemically administered genes in tumor endothelium for cancer gene therapy.

Cationic lipid-based delivery system for systemic cancer gene therapy.

A cationic lipid-based gene delivery system composed of N-[(1-(2,3-dioleyloxy)propyl)]-N-N-N-trimethylammonium chloride and cholesterol, at a 4:1 molar ratio, was developed for systemic administration. Plasmid biodistribution and expression were characterized in syngeneic mouse tumor model squamous cell carcinoma VII cells. A reporter gene expression plasmid was used for biodistribution of plasmid and expression. The results showed that lungs and primary tumors were transfected. Fluorescence microscopy showed that fluorescent-labeled transfection complexes were passively targeted to the tumor vasculature and that the endothelial cells internalized the plasmid. Transgene expression was characterized based on duration of expression and dosing schedule. In vivo gene transfer with an interleukin-12 expression plasmid yielded protein levels in blood, lungs, and primary tumor after intravenous administration. Efficacy studies showed that 15 microg of interleukin-12 plasmid was sufficient to produce a gene-specific inhibition of primary tumor growth. These results characterize the vascularity of the tumor model, characterize the in vivo gene transfer properties of the plasmid-based gene delivery system, and show that the transgene expression level was sufficient to elicit a biological response by inhibiting tumor growth.

Temporal relation of calcium-calmodulin binding and neuronal damage after global ischemia in rats.

BACKGROUND AND PURPOSE: This study explores the temporal relation of the severity of ischemia and calcium-calmodulin binding in vulnerable and resistant brain regions in a commonly used model of global ischemia. METHODS: Immunohistochemical assay of free calmodulin unbound to calcium and light microscopic histological damage were measured in rats after 5, 10, or 20 minutes of global ischemia. RESULTS: After 24 hours of reperfusion, decreased calmodulin staining, representing increased calcium influx and calcium-calmodulin binding, correlated with increasing durations of ischemia across all brain regions. Based on a 4-point scale (4, extensive stain; 0, no staining), calmodulin staining after 5 minutes versus 10 minutes of ischemia was 3.2 versus 1.9, respectively (p less than 0.05) and after 10 minutes versus 20 minutes of ischemia was 1.9 versus 1.0, respectively (p less than 0.01). The CA1 region displayed the greatest sensitivity to ischemia. Similar but less dramatic results were seen after 2 hours of reperfusion. After 72 hours of reperfusion, histological damage closely correlated with calcium-calmodulin binding after variable durations of ischemia. A threshold of 10 minutes of ischemia was required to cause calcium-calmodulin binding and irreversible neuronal damage. Surviving neuronal populations showed recovery of calmodulin staining 7 days after ischemia, representing a return of free calmodulin and normal calcium homeostasis. CONCLUSIONS: These correlations between calcium-calmodulin binding, histological damage, and duration of ischemia support the causal role of calcium influx in global ischemic injury and suggest the need for very rapid intervention after ischemia if calcium-mediated damage is to be prevented.

Neuronal protection correlates with prevention of calcium-calmodulin binding in rats.

We correlated the efficacy of several clinically relevant pharmacotherapies with their ability to prevent calcium influx into neurons and subsequent binding to calmodulin. We studied the administration of CGS 19755, nimodipine, nicardipine, and combinations of these drugs before or immediately after ischemia in globally ischemic rats. Calcium-calmodulin binding was graded by an immunohistochemical assay after 2 and 24 hours of reperfusion (n = 5-6 at each time period), and histologic damage was graded by light microscopy after 72 hours of reperfusion (n = 6). Calcium-calmodulin binding correlated with the severity of delayed histologic damage in various brain regions. In untreated ischemic control rats, marked calcium-calmodulin binding was seen in CA1 and CA3 after 24 hours of reperfusion (p less than or equal to 0.01). Administered before ischemia, CGS 19755 prevented calcium-calmodulin binding across all brain regions after 2 and 24 hours of reperfusion compared with controls (p less than or equal to 0.05). This effect was most prominent in CA3 and CA1, where the drug also reduced delayed neuronal damage (p less than or equal to 0.05). Lower doses or postischemic administration of CGS 19755, nimodipine, nicardipine, and a combination of postischemic CGS 19755 and nicardipine had a more limited effect on calcium-calmodulin binding and did not protect against delayed neuronal damage.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium-calmodulin binding in ischemic rat neurons after calcium channel blocker therapy.

Calcium channel blockers such as nicardipine improve outcome after global cerebral ischemia and may attenuate ischemic neuronal injury by preventing calcium influx and binding to calmodulin. We followed the temporal and regional sequence of neuronal calcium-calmodulin binding in normal rats (n = 6), untreated ischemic rats (n = 15), and ischemic rats treated with 0.05 mg/kg/hr s.c. nicardipine (n = 13). After 30 minutes of four-vessel occlusion, 40-microns brain sections were incubated in an anti-calmodulin antibody specific for calmodulin not bound to calcium and brain protein. Light-microscopic sections were examined immediately after ischemia and after 2 and 24 hours of reperfusion. Extensive staining of unbound calmodulin was seen in all hippocampal regions and in the cortex in normal rats. In untreated ischemic control rats, staining was lost, indicating calcium-calmodulin binding immediately after ischemia in all regions. However, after 24 hours, staining returned to normal in the cortex and dentate, and minimal staining returned in CA1 and CA3. Nicardipine-treated animals had significantly less calcium-calmodulin binding in CA1 and in the dentate after 2 hours of reperfusion. This study demonstrates that in clinically relevant doses nicardipine has a limited effect on calcium-calmodulin binding in selectively vulnerable regions after severe ischemia.

CGS-19755, a competitive NMDA receptor antagonist, reduces calcium-calmodulin binding and improves outcome after global cerebral ischemia.

We evaluated several doses of cis-4-(phosphonomethyl)-2-piperidine-carboxylic acid (CGS-19755), a potent competitive N-methyl-D-aspartate (NMDA) receptor antagonist, systemically administered either before or after 20 to 30 minutes of global ischemia in rats. We measured outcome by mortality, histological damage by light microscopy, and learning ability on an eight-arm maze, and determined the drug's mechanism of action by an immunohistochemical assay of calcium-calmodulin binding. High-dose treatment begun prior to ischemia resulted in reduced cellular damage in severely ischemic hippocampal tissue, but also caused high mortality due to respiratory depression. Treatment begun 30 minutes after ischemia resulted in little histological protection but significantly improved learning ability when tested 1 month after ischemia, and did not increase mortality. Furthermore, CGS-19755, 10 mg/kg intraperitoneally, begun either before or after ischemia substantially reduced calcium influx into ischemic neurons as evidenced by reduced calcium-calmodulin binding. We conclude that CGS-19755 prevents calcium entry into ischemic neurons and may be effective therapy for very acute cerebral ischemia.

Immunohistochemical determination of calcium-calmodulin binding predicts neuronal damage after global ischemia.

Since ionic Ca2+ binds with intracellular calmodulin (CaM) before activating proteases, kinases, and phospholipases, demonstration of persistent Ca2+-CaM binding in neurons destined to show ischemic cellular injury would support the concept that elevated intracellular Ca2+ plays a causative role in ischemic neuronal damage. In order to characterize Ca2+-CaM binding, we used a sheep anti-CaM antibody (CaM-Ab) which recognizes CaM that is not bound to Ca2+ or brain target proteins. Therefore, immunohistochemical staining of brain sections by labeled CaM-Ab represented only unbound CaM. Six normal rats were compared to 15 animals rendered ischemic for 30 min by a modification of the four-vessel occlusion model. Animals were killed immediately after ischemia, and after 2 and 24 h of reperfusion. Brain sections through hippocampus were incubated in CaM-Ab, and a diaminobenzadiene labeled anti-sheep secondary antibody was added to stain the CaM-Ab. Staining in the endal limb of dentate, dorsal CA1, lateral CA3, and parietal cortex was graded on a 4-point scale. All normal animals had grade 4 staining indicating the presence of unbound CaM in all four brain regions. Ischemic animals demonstrated reduced (grade 0 to 2) staining in the CA1 and CA3 regions immediately and 2 and 24 h after ischemia (p less than 0.01 for both regions at all three time intervals) indicating persistent binding of CaM with Ca2+ and target proteins in these regions. Staining decreased in dentate and cortex up to 2 h after ischemia (p = 0.02 for both regions) but returned toward normal by 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Prediction of carotid stenosis progression by lipid and hematologic measurements.

We followed 19 men and 19 women with asymptomatic carotid stenosis up to 30 months to determine whether hematologic or lipid abnormalities could identify those individuals developing progressing carotid atherosclerosis (defined as an increase in mean percent stenosis greater than or equal to 19% or an increase in a single region of greater than or equal to 23%) on B-mode carotid ultrasonography performed at 2- to 6-month intervals. Our patients demonstrated increased beta-thromboglobulin, platelet factor 4, and fibrinogen compared with age-matched controls. Eight patients developed progression of carotid stenosis, and this group had higher baseline low-density lipoprotein (LDL) and fibrinogen than the 30 nonprogressing patients. Multiple regression analyses of age, sex, smoking, coronary artery disease, peripheral vascular disease, diabetes, hypertension, and baseline high-density lipoprotein (HDL), HDL2, HDL3, LDL, beta-thromboglobulin, platelet factor 4, and fibrinogen identified coronary artery disease and elevated LDL and fibrinogen as the only independent variables significantly associated with the progressing group. We conclude that, in patients with carotid atherosclerosis, a combination of coronary artery disease and elevated LDL and fibrinogen will predict with 88% accuracy whether the patient will have progressing carotid stenosis.

Identification and subcellular localization of a 21-kilodalton molecule using affinity-purified antibodies against alpha-transforming growth factor.

Monospecific antibodies were generated against each of six different peptide sequences derived from rat and human alpha-transforming growth factor (alpha-TGF). The affinity-purified antibody to the 17 amino acid carboxyl-terminal portion of the molecule proved most useful in detecting alpha-TGF. When used in a peptide-based radioimmunoassay, it was possible to measure nanogram quantities of native alpha-TGF in conditioned cell culture media. When used to analyze cell lysate, these antibodies specifically recognized a 21-kilodalton protein species. Indirect immunofluorescence localization procedures revealed a high concentration of alpha-TGF in a perinuclear ring with a diffuse cytoplasmic distribution. These results suggest that a precursor form of alpha-TGF has a cellular role beyond that of an autocrine growth factor.