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V V Kakkar

Publications and source records attributed to V V Kakkar.

At least 37 records · Page 2Linked to original sources
PubMed1999

Design of a muscle cell-specific expression vector utilising human vascular smooth muscle alpha-actin regulatory elements.

The facility to direct tissue-specific expression of therapeutic gene constructs is desirable for many gene therapy applications. We describe the creation of a muscle-selective expression vector which supports transcription in vascular smooth muscle, cardiac muscle and skeletal muscle, while it is essentially silent in other cell types such as endothelial cells, hepatocytes and fibroblasts. Specific transcriptional regulatory elements have been identified in the human vascular smooth muscle cell (VSMC) alpha-actin gene, and used to create an expression vector which directs the expression of genes in cis to muscle cells. The vector contains an enhancer element we have identified in the 5' flanking region of the human VSMC alpha-actin gene involved in mediating VSMC expression. Heterologous pairing experiments have shown that the enhancer does not interact with the basal transcription complex recruited at the minimal SV40 early promoter. Such a vector has direct application in the modulation of VSMC proliferation associated with intimal hyperplasia/restenosis.

Actins↗
PubMed1999

Tissue-selective expression of dominant-negative proteins for the regulation of vascular smooth muscle cell proliferation.

The transcription factors c-myb and c-myc are essential for vascular smooth muscle cell (VSMC) replication and are rapidly induced following mitogenic stimulation of quiescent VSMCs in vitro and in vivo following balloon catheter injury. Consequently, interference with c-myb and c-myc function provides a possible avenue for the prevention of VSMC proliferation associated with intimal hyperplasia. We have carried out studies focused on the inhibition of VSMC proliferation using dominant-negative gene constructs incorporating the DNA-binding domains of the c-myb or c-myc genes fused to the repressor domain of the Drosophila engrailed gene. Transient transfection of rat, rabbit and human vascular SMCs results in a dramatic inhibition of proliferation for at least 72 h after transfection. Furthermore, this inhibition of cellular proliferation was found to be due, at least in part, to the induction of apoptosis. Coupling expression of the chimeric dominant-negative proteins to transcriptional regulatory elements of the human vascular smooth muscle alpha-actin gene allows specific targeting of vascular smooth muscle cells.

Actins↗
PubMed1999

Haemodynamic responses and changes of haemostatic risk factors in cold-adapted humans.

Epidemiological studies have shown an increase in acute myocardial infarctions or deaths due to myocardial infarction in colder weather; the mechanisms most likely involve increased blood levels of haemostatic risk factors, and increases in arterial blood pressure and heart rate. We studied the relationship between cold adaptation, haemostatic risk factors and haemodynamic variables. Cold adaptation was obtained by a programme of immersion of the whole body up to the neck in a water-filled bath, the temperature of which was gradually decreased from 22 degrees C to 14 degrees C, time of exposure being increased from 5 to 20 min over a period of 90 days. We studied 428 patients (44% men) and measured blood levels of fibrinogen, plasminogen activator inhibitor 1 (PAI-1), tissue plasminogen activator antigen (t-PA), plasma viscosity, von Willebrand factor, D-dimer and platelet count, both at baseline and after 90 days of daily immersion. There were significant reductions in von Willebrand factor (-3%; p < 0.001), and plasma viscosity (-3.0 s; p < 0.001), and a mild but significant increase in PAI-1 (+0.3 IU/ml; p = 0.02). The pressure rate product (systolic blood pressure x heart rate) was also significantly lower after cold adaptation (-310; p = 0.004). Cold adaptation, compared with exposure to cold weather, induces different haemodynamic responses and changes of blood levels of haemostatic risk factors.

Acclimatization↗
PubMed1999

A cyclic peptide analogue of the loop III region of platelet-derived growth factor-BB is a synthetic antigen for the native protein.

We report the synthesis and characterization of a cyclic peptide analogue of the loop III region of platelet-derived growth factor (PDGF) B-chain sequence, cyclo(73Arg-Lys-Ile-Glu-Ile-Val-Arg-Lys-Lys81-Cys), incorporating a C-terminus cysteine residue for the conjugation to a carrier protein. The synthesis involved solid-phase chemistry, utilizing Fmoc-tBu chemistry and acid labile side-chain protecting groups, followed by 'head-to-tail' cyclization using the allyl-protected glutamic acid anchored on its side chain to the solid support with HATU/HOAt as the coupling agent. Conformational differences between the cyclic and its linear counterpart PDGF peptides were determined by circular dichroism measurements in aqueous media. High titre antisera were raised to both cyclic and linear peptide immunogens. Antisera raised to the cyclic peptide cross-reacted with PDGF-BB in both Western blot and ELISA, whereas antisera raised to the linear peptide had no reactivity with PDGF-BB. The cyclic peptide (conformational design analogue) produces an immunogen which is able to antigenically mimic the secondary structure of loop III of PDGF-BB and forms a basis from which further small molecular mimetics of PDGF may be designed for use as both immunogens and also potential agonists/antagonists of PDGF. Similarly constructed immunogens may also be useful in the design of vaccines which direct responses to loop regions in other target proteins.

Animals↗
PubMed1999

Cellular effects of heparin on the production and release of tissue factor pathway inhibitor in human endothelial cells in culture.

Tissue factor pathway inhibitor (TFPI), the major downregulator of procoagulant activity of the tissue factor-factor VIIa complex (TF. FVIIa), is synthesized and constitutively secreted by endothelial cells (ECs). Here we describe the in vitro effects of heparin on the cellular localization, gene expression, and release of TFPI in human ECs in culture. Both unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH; Fragmin) time-dependently induced a significant enhanced secretion of TFPI, paralleled by a redistribution and increase of TFPI on the cell surface and a decrease of intracellular TFPI. Immunogold electron microscopy showed the presence of clusters of TFPI, both on the plasmalemma proper and within cell-surface opened caveolae/enlarged caveolar profiles. Activation of FX by TF. FVIIa on ECs treated with endotoxin was inhibited by both heparins but to a higher extent by LMWH. Inhibition of protein synthesis by cycloheximide did not reduce the release of TFPI induced by heparin. Long-term incubation (48 hours) resulted in a time-dependent enhanced production of TFPI. After the first 4 to 8 hours, depletion of intracellular TFPI was observed, more significantly with UFH. Northern blot analysis of TFPI mRNA also showed a decrease of the 1.4-kb transcript after 4 hours of incubation with UFH, followed by recovery and an increase over the control level after 24 hours. Incubation of ECs with phorbol ester (PMA) significantly enhanced the secretion of TFPI and increased its activity on the cell surface, probably by preventing invagination of caveolae. Heparin-stimulated release of TFPI decreased significantly in the presence of PMA to a level that was 2. 4 times lower than the expected additive value for PMA and UFH separately. Pretreatment of ECs with PMA suppressed a subsequent response to heparin. Altogether, our results suggest that the heparin-induced release of TFPI might involve a more specific mechanism(s) than the previously hypothesized simple displacement of TFPI from the cell surface glycocalyx. We assume that the increased secretion and redistribution of cellular TFPI induced by heparins in ECs in culture can play an important role in the modulation of the anticoagulant properties of the endothelium.

Cell Line↗
PubMed1999

The significance of creatine kinase in cardiac patients with acute limb ischaemia.

OBJECTIVE: The value of creatine kinase (CK) and aspartate transaminase (AST) has not been previously evaluated following cardiac surgery in the diagnosis of acute limb ischaemia. Our objective was to assess the value of CK and AST with reference to the diagnosis of limb ischaemia, effect on renal function and prognosis following cardiac surgery. DESIGN: all patients entering ICU had daily CK and AST measurements over a two -year period. A retrospective study of patients with CK values >5,000 U/L was performed. SETTING: adult intensive care unit of a secondary and tertiary referral centre for cardiothoracic surgery with on site facilities for vascular surgery. PATIENT: twenty-seven patients had CK values greater than 5,000 U/L. A further random sample of 35 patients, with no limb ischaemia were used to give medians for CK and AST following cardiac surgery. INTERVENTIONS: twelve of twenty-seven (44%) patients were noted to have acutely ischaemic limbs; 6 of these (CK>16,000 U/L) underwent surgical intervention. MEASURES: serum levels of CK, Peak CK, AST, Peak AST and CK/AST ratios. Related to procedure and outcome in terms of mortality and morbidity including, acute limb ischaemia defined on clinical grounds and renal failure defined as creatinine >200 pmoles/L. RESULTS: The median values for CK and AST immediately following uncomplicated cardiac surgery were 135 (IQR 36-383) and 43 (IQR 26-58) respectively. Median CK for the patients (CK>5,000) without clinical ischaemia was 7,440 U/L compared to the group with ischaemia 17,472 U/L (p<0.05). Renal failure developed in 48% of all patients. Eight of the 9 patients with CK>15,000 developed renal failure compared to 5 of the 13 with CK 5,000-15,000 U/L (p<0.01). 30% of patients underwent haemofiltration; of these, 87% died. For patients with ischaemia peak CK and creatinine correlated. (r = 0.83, p<0.05) Day of peak CK and peak AST correlated (r = 0.92, p<0.01). Logged values of CK with log values of AST showed a highly significant relationship (beta = 1.16, p<0.001). The overall mortality was 33%. CONCLUSIONS: PATIENTs who develop limb ischaemia following cardiac surgery have a high morbidity and mortality. A CK of 17,000 U/L (5667-46539) is indicative of serious limb ischaemia. Renal failure is likely to develop in patients with CK>15,000 U/L. AST may become a useful additional marker of limb ischaemia.

Acute Disease↗
PubMed1999

Enhancement of lipoprotein lipase activity by tissue factor pathway inhibitor.

The effect of a basic synthetic peptide, representing the C-terminal region of tissue factor pathway inhibitor (TFPI - Lys254 - Met276), as well as that of the whole protein, on the activity of lipoprotein lipase (LPL) is described. The activity of bovine LPL was measured by chromogenic assay using a water-soluble chromogenic substrate, p-nitrophenyl butyrate. Five and 10 microM concentrations of the peptide increased Vmax of bovine LPL by 48.9% and 85.6% respectively as compared with the buffer control without affecting Km. Poly l-lysine, though positively charged did not have any effect, suggesting the importance of the amino acid sequence of the test peptide. On the other hand, 0.25, 0.5 and 1.0 mM n-butyric acid - a product of LPL catalysis in the chromogenic assay, when added to the incubation mixture decreased Vmax non competitively by 22.8%, 40.4% and 63% respectively as compared with buffer control, confirming the known product inhibition of LPL. A 100-fold molar excess of n-butyric acid produced inhibition of the LPL reaction as compared with the synthetic peptide which produced potentiation, suggesting a 1:100 stoichiometric interaction of the peptide with n-butyric acid. At a fixed concentration of 0.25 mM substrate, 10 nM full length recombinant TFPI, containing the basic C-terminal domain, increased velocity of LPL reaction by 39.4% as compared with buffer control. The same concentration of two-domain recombinant TFPI (TFPI1-160) had no effect. It is possible that negatively charged n-butyric acid is sequestered by the positively charged peptide or the basic region of recombinant full length TFPI. Relieving of product inhibition could then be a possible mechanism of the observed potentiation of bovine LPL activity by the basic peptide or full length recombinant TFPI. The 39.4% increase in reaction velocity of LPL catalysis produced by 10 nM full length recombinant TFPI was comparable to 38.9% increase produced by 5 microM of the basic peptide under the same conditions. A further increase of 78.7% was brought about by 10 microM concentration of the same peptide. The reason for about 500-fold increase in the potency of the whole protein as compared with that of the peptide is not clear. It is possible that in its tertiary conformational state, the whole protein is able to sequester product and relieve product inhibition more effectively than the short linear peptide. Rabbit polyclonal antiserum against the basic peptide partially inhibited LPL activity of human post heparin plasma, measured by radioenzymatic assay using triolein substrate. Since post heparin plasma contains full length TFPI, binding of the added antibody to its basic C-terminus and hence the relative unavailability of latter for product sequestration (oleic acid in this case) could explain the observed inhibition of human LPL activity by antibody against the peptide. Thus by enhancing lipase activity, full length TFPI may facilitate hydrolysis of triglyceride and concomitantly lower factor VII coagulant activity as demonstrated earlier, particularly after heparin injection when both TFPI and LPL are released in circulation.

Amino Acid Sequence↗
PubMed1999

Acute release of tissue factor pathway inhibitor after in vivo thrombin generation in baboons.

Tissue factor pathway inhibitor (TFPI), the major downregulator of the procoagulant activity of tissue factor (TF), is synthesised by endothelial cells (EC) and acutely released in vitro after thrombin stimulation. Expression of TF on EC and subsequent thrombin generation occurs in vivo during sepsis or malignancy, inducing disseminated intravascular coagulation (DIC). The present study investigates the changes in plasma TFPI in relation to markers of in vivo thrombin generation induced by injection of factor Xa (FXa)/phospholipids in baboons at dosages leading to partial (48%) or complete fibrinogen depletion. The plasma concentrations of thrombin-antithrombin III (TAT) and fibrinopeptide A (FpA), as markers of in vivo generation of thrombin, were strongly enhanced after injection of FXa/phospholipids. TFPI levels, whether measured as antigen or activity, increased significantly in both treatment groups within few minutes, and were dependent on the dose of FXa/phospholipids. Significant positive correlations between plasma levels of TFPI and of TAT or FpA were observed. Altogether, our results indicate that experimentally induced in vivo generation of thrombin causes the acute release of TFPI, high-lighting a possible novel function of thrombin in downregulation of the coagulation process, potentially relevant for the outcome of DIC.

Animals↗
PubMed1999

Toward a rational management of concomitant carotid and coronary artery disease.

BACKGROUND: Patients with angina undergoing carotid endarterectomy have a high perioperative mortality. Our aim was to assess the outcome of carotid endarterectomy in patients with concomitant coronary artery disease, in particular, to examine the timing of carotid endarterectomy and CABG. METHODS: A retrospective study was performed at a tertiary and secondary referral centre for cardiovascular disease. Over a five-year period 71 carotid endarterectomies and 6,590 coronary artery bypasses were performed. Significant (>70%) internal carotid stenoses were found in 35 (0.5%) patients due for CABG. Of these, 23 patients underwent Combined procedures (Carotid and CABG), 9 Reverse staged (CABG then Carotid), and 3 Prior staged carotid endarterectomies (Carotid then CABG). 36 other carotid endarterectomies were in patients evaluated cardiologically, but did not require CABG (Isolated group). Risk factors, 30 day perioperative outcome including hospital inpatient stay and early follow-up are reported. RESULTS: The Combined group 30-day perioperative mortality rate was 4.3% and permanent stroke rate 8.6%. There were no major complications in Reverse or Prior staged cases. Isolated group mortality was 2.7% with no strokes. Risk factors were more prevalent in the combined group; 56% previous myocardial infarction, 39% hypertension, 35% a history of raised cholesterol and 46% intermittent claudication. All cases were followed up for a mean of 18.4 months, with no carotid stroke related events. Overall hospital stay for staged patients was a mean 19.3 days (SE=2.4) days compared to mean 9.8 days for combined patients (SE=0.97, p<0.001). CONCLUSIONS: Patients with combined cardiac and carotid disease benefit from assessment of both systems in order to stage CABG and carotid endarterectomy. Risk factors were more prevalent in the combined group; a combined procedure offered a median difference of 8 days less hospital inpatient stay compared to the staged cases. Our experience suggests that carefully planned management of concomitant coronary and carotid disease can achieve better results.

Aged↗
PubMed1998

Bifunctional peptide boronate inhibitors of thrombin: crystallographic analysis of inhibition enhanced by linkage to an exosite 1 binding peptide.

The affinity of the hirudin49-64 segment for exosite 1 of thrombin has been used previously to enhance the potency of simple competitive inhibitors [DiMaio, J., Gibbs, B., Munn, D., Lefebvre, J. , Ni, F., Konishi, Y. (1990) J. Biol. Chem. 265, 21698-21703., and Maraganore, J. M., Bourdon, P., Jablonski, J., Ramachandran, K. L., and Fenton, J. W., II (1990) Biochemistry 29, 7095-7087.]. Using a similar approach, we have enhanced the activity of two active site directed thrombin inhibitors by attaching this segment via a novel reverse oriented linker to each of two tripeptide boronate inhibitors. At P1, compound 1 contains an arginine-like, isothiouronium, side chain, while compound 2 contains an uncharged, bromopropyl residue. Inhibition of human alpha-thrombin by compound 1 shows slow, tight-binding competitive kinetics (final Ki of 2.2 pM, k1 of 3.51 x 10(7) M-1 s-1, and k-1 of 1.81 x 10(-)4 s-1). The addition of hirugen peptide (20 microM) competes for exosite 1 binding and restores the k1 and k-1 to that of the analogous tripeptide, 0.29 x 10(7) M-1 s-1 and 0.13 x 10(-)4 s-1, respectively. Compound 1 has enhanced specificity for thrombin over trypsin with KiTry/KiThr of approximately 900 compared to the analogous tripeptide, with KiTry/KiThr of approximately 4. Compound 2 acts as a competitive inhibitor (KiThr of 0.6 nM) and is highly selective with no effect on trypsin. Crystallographic analysis of complexes of human alpha-thrombin with compound 1 (1.8 A) and compound 2 (1.85 A) shows a covalent bond between the boron of the inhibitor and Ser195 (bond lengths B-O of 1.55 and 1.61 A, respectively). The isothiouronium group of compound 1 forms bidentate interactions with Asp189. The P2 and P3 residues of the inhibitors form interactions with the S2 and S3 sites of thrombin similar to other D-Phe-Pro based inhibitors [Bode, W., Turk, D., and Karshikov, A. (1992) Protein Sci. 1, 426-471.]. The linker exits the active site cleft of thrombin forming no interactions, while the binding of Hir49-64 segment to exosite 1 is similar to that previously described for hirudin [Rydel, T. J., Tulinsky, A., and Bode, W. (1991) J. Mol. Biol. 221, 583-601.]. Because of the similarity of binding at each of these sites to that of the analogous peptides added alone, this approach may be used to improve the inhibitory activity of all types of active site directed thrombin inhibitors and may also be applicable to the design of inhibitors of other proteases.

Amino Acid Sequence↗
PubMed1998

Prevention of venous thromboembolism in general surgery.

Venous thromboembolism continues to be an important cause of death in hospitalized patients undergoing major elective surgery. A study of autopsy-proven pulmonary embolism in hospital patients showed that venous thromboembolism accounted for 10% of deaths and that recognition of non-fatal thromboembolism continues to be a problem. The incidence of deep vein thrombosis increases with ageing, the annual rate per 1000 being one to three for those aged between 65 and 69 years and from two to eight for those aged between 85 and 89 years. The introduction of low-molecular-weight heparins has resulted in important changes in the management and prophylaxis of venous thromboembolism. Low-molecular-weight heparin preparations reduce the overall incidence of deep vein thrombosis in general surgery by at least 70%. Furthermore, the effect of low-molecular-weight heparin against pulmonary embolism is at least as great as that of low-dose unfractionated heparin. The incidence of serious and minor haemorrhagic events with low-molecular-weight heparin is similar to that with low-dose unfractionated heparin. Prophylaxis is started pre-operatively, and the usual duration for the post-operative period has been 7 days, or until the patient is discharged from the hospital. In conclusion, low-molecular-weight heparin is highly effective in preventing post-operative venous thromboembolism.

Aged↗
PubMed1998

Chronic fatigue syndrome: physical and cardiovascular deconditioning.

We investigated whether chronic fatigue syndrome (CFS) patients have physical and/or cardiovascular de-conditioning, in 273 CFS patients and 72 healthy controls. We used laboratory tests to assess haematological, biochemical, endocrinological and immunological systems. The cardiovascular system was assessed by echocardiography and carotid echography. Body composition was determined by dual energy X-ray absorptiometry (DEXA). CFS patients had smaller left ventricular end systolic (p < 0.001) and diastolic (p = 0.008) dimensions but thinner posterior walls (p = 0.02) than corresponding values in healthy controls. Left ventricular mass was also reduced in CFS patients (p = 0.006). Both maximum (p < 0.001) and minimum (p < 0.008) diameter of the carotid artery were smaller in CFS patients. The laboratory screening tests showed significant differences in serum albumin (p = 0.05), phosphate (p = 0.02), HDL-cholesterol (p = 0.03), HDL:total cholesterol ratio (p = 0.01), triglycerides (p = 0.02), neutrophils (p = 0.01) and thyroid-stimulating hormone (p = 0.04) between CFS patients and controls. Male CFS patients had an increased percentage of fat mass compared with healthy male subjects (p = 0.02). This large group of CFS patients had evidence of physical and cardiovascular de-conditioning, suggesting that in these patients a graded exercise programme could lead to physical reconditioning and could increase their ability to perform physical activities.

Adult↗