Kanti D Bhoola

Kinin receptors in human vascular tissue: their role in atheromatous disease.

Using samples of many human blood vessels, obtained at autopsy and specific antibodies directed to peptide sequences of the kinin B1 and B2 receptors, we demonstrate the localisation of these receptors within the human vascular system using standard immunolabelling techniques. In large elastic arteries and veins, kinin receptors are present only in the endothelial cells whereas in all muscular arteries and arterioles, these receptors are present in both the endothelial and smooth muscle cells. The identification of kinin receptors in human blood vessels confirms that kinins may modulate both vascular permeability and contractility. The incidental finding at histology, of patchy atheromatous disease in the coronary, femoral, vertebral and pericallosal arteries, assisted in elucidating the role of these receptors in the commonest disease affecting human blood vessels. Intense labelling for B1 receptors was observed in the endothelial cells, foamy macrophages, inflammatory cells and fibroblasts within the thickened intima of the plaque as well as in smooth muscle cells of the underlying tunica media. Immunoreactive B2 receptors were also observed in these cells but with reduced intensity. The intense immunolabelling of B1 receptors in these regions suggest that these may be induced by atheromatous disease and may have therapeutic importance for the B1 receptor antagonists.

Experimental evaluation of collagen sponge as a dural graft

Early collagen products, when used as dural substitutes, promoted severe inflammatory responses and fell into disrepute. A more recent advance, collagen sponge, which is derived from bovine flexor tendons was used in this experimental study. Collagen sponge was surgically implanted as an onlay dural replacement graft following skull trephination and dural excision in 12 primates. Macroscopic, histological and electron-microscopical evaluations were performed at periods of 1, 3 and 9 months. This preliminary animal study indicated that collagen sponge is suitable to use as a graft since it does not induce any inflammatory response or adhesions in the absence of pia arachnoid injury. If forms an ideal scaffold for the early ingrowth of fibroblasts to effect dural repair.

Kinin receptors on human neurones

Knowledge of the distribution of kinin receptors in the human brain will aid our understanding of the role of kinins in neurophysiology. Furthermore, induction of the kinin B1 receptor may be important in the pathogenesis of neural diseases. Using polyclonal antibodies directed to specific regions of the B1 and B2 kinin receptors and standard immunolabelling techniques, we report on the localisation of these receptors on neurones in specific areas of the human brain. B2 bradykinin receptors are present in neurones of the brain stem, basal nuclei, cerebral cortex, thalamus and hypothalamus. B2 immunolabelling was also observed in the endothelial lining of the superior sagittal dural sinus and ependyma of the lateral and third ventricles. B1 kinin receptors have been localised on neurones of the thalamus, spinal cord and hypothalamus. Although binding of labelled bradykinin to neuronal membranes has been demonstrated, this is the first conclusive evidence for the existence of immunoreactive B1, and further confirmation of B2 receptors on human neurones.

PATHOPHYSIOLOGY OF THE KALLIKREIN-KININ SYSTEM IN MAMMALIAN NERVOUS TISSUE

The nervous system and peripheral tissues in mammals contain a large number of biologically active peptides and proteases that function as neurotransmitters or neuromodulators in the nervous system, as hormones or cellular mediators in peripheral tissue, and play a role in human neurological diseases. The existence and possible functional relevance of bradykinin and kallidin (the peptides), kallikreins (the proteolytic enzymes), and kininases (the peptidases) in neurophysiology and neuropathological states are discussed in this review. Tissue kallikrein, the major cellular kinin-generating enzyme, has been localised in various areas of the mammalian brain. Functionally, it may assist also in the normal turnover of brain proteins and the processing of peptide-hormones, neurotransmitters, and some of the nerve growth factors that are essential for normal neuronal function and synaptic transmission. A specific class of kininases, peptidases responsible for the rapid degradation of kinins, is considered to be identical to enkephalinase A. Additionally, kinins are known to mediate inflammation, a cardinal feature of which is pain, and the clearest evidence for a primary neuronal role exists so far in the activation by kinins of peripherally located nociceptive receptors on C-fibre terminals that transmit and modulate pain perception. Kinins are also important in vascular homeostasis, the release of excitatory amino acid neurotransmitters, and the modulation of cerebral cellular immunity. The two kinin receptors, B2 and B1, that modulate the cellular actions of kinins have been demonstrated in animal neural tissue, neural cells in culture, and various areas of the human brain. Their localisation in glial tissue and neural centres, important in the regulation of cardiovascular homeostasis and nociception, suggests that the kinin system may play a functional role in the nervous system.

Expression of tissue kallikrein and kinin receptors in angiogenic microvascular endothelial cells.

Abstract Angiogenesis is the sprouting of new capillary blood vessels from pre-existing ones. The kinin family of vasoactive peptides, formed by the serine protease tissue kallikrein from its endogenous multifunctional protein substrate kininogen, is believed to regulate the angiogenic process. The aim of this study was to determine the expression of tissue kallikrein and kinin receptors in an in vitro model of angiogenesis. Microvascular endothelial cells from the bovine mature and regressing corpus luteum were used only if they reacted with known endothelial cell markers. At first the cultured endothelial cells began sprouting, and within four weeks formed three-dimensional, capillary-like structures. Immunolabelling for tissue prokallikrein and the mature enzyme was intense in the angiogenic endothelial cells derived from mature corpora lutea. Immunoreactivity was lower in non-angiogenic endothelial cells and least in angiogenic endothelial cultures of the regressing corpus luteum. Additionally, using specific antisense DIG-labelled probes, tissue kallikrein mRNA was demonstrated in cells of the angiogenic phenotype. Immunolabelled kinin B2 receptors, but not kinin B1 receptors, were visualised on angiogenic endothelial cells. Our results suggest an important regulatory role for kinins in the multiple steps of the angiogenic cascade that may occur in wound healing and cancer cell growth.

Hyponatremic natriuretic syndrome in tuberculous meningitis: the probable role of atrial natriuretic peptide.

Hyponatremia has been reported in up to one third of patients with intracranial disease and has frequently been associated with tuberculous meningitis, often complicated by hydrocephalus. The lowered plasma sodium levels were previously attributed to the syndrome of inappropriate secretion of antidiuretic hormone. A controlled prospective study of 24 patients with tuberculous meningitis and hydrocephalus was carried out. Analyses of serum electrolytes and cerebrospinal fluid were performed. Plasma and cerebrospinal fluid levels of atrial natriuretic peptide (ANP) and antidiuretic hormone (ADH) were measured by radioimmunoassay. Fifteen patients were found to be hyponatremic (plasma sodium < 130 mmol/L) and ANP levels of 12 to 1,488 pg/ml were present (median, 26 pg/ml). The remaining 9 patients had normal plasma sodium values between 130 and 145 mmol/L, and in these, plasma ANP values varied between 12 and 21.7 pg/ml (median, 12 pg/ml). The difference between these two groups was not statistically significant. (Control values from patients undergoing myelography were established to range between 12 and 40 pg/ml; median, 14.4 pg/ml.) ANP levels were undetectable in the cerebrospinal fluid in all. Plasma ADH levels in the hyponatremic group were between 7 and 159 pg/ml (median, 40 pg/ml). In the normonatremic group, plasma ADH levels of 25 to 250 pg/ml (median, 29 pg/ml) were obtained. (The controls ranged between 3.6 and 35 pg/ml; median, 10.4 pg/ml). In the hyponatremic group, there was a moderate negative correlation (r = -0.683) between plasma ANP and plasma sodium (P = 0.02). No correlation between plasma ADH and plasma sodium was found (r = -0.168; P = 0.62).(ABSTRACT TRUNCATED AT 250 WORDS)

Kinin receptors are expressed in human astrocytic tumour cells.

Tissue kallikrein (TK) is known to be present in several tumours in which increased KLK1 (TK) gene expression has been demonstrated. By degrading components of the extracellular matrix, TK may facilitate tumour proliferation and invasion. The vasodilatory effect of the bioactive kinin peptides causes an increase in vascular permeability, thereby enhancing metastasis. Since kinins act by receptor-linked signal transduction mechanisms, the aim of this study was to elucidate the localization and expression of kinin B1 and B2 receptors in surgical samples of human astrocytic tumours. Tumour tissue collected was processed for light, confocal and electron microscopy (EM) and RNA extraction. The mean high intensity of immunolabeling in tumour cells was quantified in pixels per square micrometer using the Analysis 2.1 Prosystem (Soft-Imaging Software, Germany, 1996). The ultrastructural localization of B1 and B2 kinin receptors was performed on ultrathin sections of the resin-embedded tissue, using immunogold-labeled probes. In the human brain, immunoreactive B2 occurs in cortical neurones but not in glial cells, and immunolabeling for B1 receptors is absent in cortical areas. In the present study, in all of the tumours studied so far, immunolabeling for B2 (28.42 pixels/microm2, n = 12) and B1 (14.07 pixels/ microm2, n = 10) was observed on the astrocytic cells. Immunoreactive kinin receptors were also present in endothelial cells of the stromal blood vessels. At EM, the average number of immunogold particles was 14 for B2 receptors and eight for B1 receptors. The immunoreactive B2 receptors were located closer to the periphery of the tumour cells while B1 immunolabeling was observed throughout the cell.

Tissue kallikrein and kinins in renal disease

The renal kallikrein-kinin system is involved in sodium and water homeostasis, blood pressure regulation and inflammation. Tissue kallikrein and kinin levels were measured in the urine of patients with renal disease and in the urine of living related kidney donors prior to uninephrectomy who served as controls. Tissue kallikrein and kinin B1 and B2 receptors were immunolocalised by confocal microscopy in renal biopsy material from patients with renal disease and controls (fresh autopsy material and normal kidney tissue from nephrectomies for malignancy). Urinary tissue kallikrein excretion was significantly decreased in patients with mild renal disease (16.6 +/- 6.7 ng tissue kallikrein (TK)/ng protein; p < 0.05) and more markedly so (1.8 +/- 0.7 ng TK/microg protein; p < 0.01) in patients with severe renal failure requiring dialysis compared to normal controls (78.9 +/- 31.7 ng TK/microg protein). Basal kinin values were unchanged in patients with renal disease (14 +/- 0.8 ng/ml) compared to controls (13.3 +/- 0.56 ng/ml). In control kidney tissue kallikrein was immunolocalised in the distal connecting tubules and collecting ducts whereas decreased immunolabelling was observed with renal disease. Kinin B2 receptor labelling was present in the entire nephron in the normal control kidney but was reduced with renal disease. While kinin B1 receptor immunolabelling was not observed in the control kidneys, labelling of distal tubules and collecting ducts was noted in renal disease, suggesting an upregulation of B1 receptors in renal parenchymal disease.

Traumatic Brain Contusions: A Clinical Role for the Kinin Antagonist CP-0127

Summaryu2003Focal cerebral contusions can be dynamic and expansive, leading to delayed neurological deterioration. Due to the high mortality associated with such cerebral contusions, our standard practice had evolved into evacuating contusions in patients who had a deterioration in level of consciousness, lesions>30 cc and CT suggestion of raised ICP. Experimental brain edema studies have implicated kinins in causing 2° brain swelling. CP-0127 (Bradycor TM), a specific bradykinin antagonist, has been found to reduce cerebral edema in a cold lesion model in rats. In a randomized, single blind pilot study, a 7 day infusion of CP-0127 (3.0 ug/kg/min) was compared to placebo in patients with focal cerebral contusions presenting within 24–96 hours of closed head injury with an initial GCS 9–14. The ICP, GCS, and vital signs were monitored hourly. The total lesion burden (TLB) was measured on serial CT scans.u2003There were no differences in age, baseline GCS, TLB, initial ICP, or laboratory findings between the two groups (n=20). The mean (±s.d) rise in peak ICP from baseline was greater in the placebo group than with CP-0127 (21.9±4.7 vs 9.5±2.0, P=0.018). In addition, the mean reduction in GCS in the placebo group was significantly greater than in the CP-0127 group (4±1.0 vs 0.6±0.4, P=0.002). Significantly raised ICP and clinically significant neurological deterioration occurred in 7/9 patients on placebo (77%) and only in 1 patient (9%; n=11) on CP-0127, mandating surgery (P=0.005). There were no adverse drug reactions, significant changes in vital signs or variations in the laboratory values. The cerebral perfusion pressure was adequately maintained in all patients irrespective of therapy.u2003These preliminary results with CP-0127 provide supporting evidence that the kinin-kallikrein system could be involved in cerebral edema. In this study, treatment with CP-0127 appeared to alter the natural history of traumatic brain contusions by preventing the 2° brain swelling. In addition, CP-0127 obviated the need for surgery in the majority of treated patients. CP-0127 could act on the cerebral vasculature to limit dys-autoregulation and brain swelling or on the blood brain barrier to reduce cerebral edema.

Upregulation of tissue kallikrein, kinin B1 receptor, and kinin B2 receptor in mast and giant cells infiltrating oesophageal squamous cell carcinoma

Background: The mitogenic kinin peptides formed by the serine protease, tissue kallikrein (TK1), stimulate the proliferation of tumour cells and, by increasing vascular permeability, enhance metastasis. Oesophageal mucosal epithelial cells are derived from the epithelial cell germ layer, which expresses the kallikrein–kinin cascade. Aim: To determine the cellular distribution of active TK1, prokallikrein, and the kinin B1 and B2 receptors in oesophageal carcinoma by immunocytochemistry and in situ hybridisation (ISH). Methods: Fifty oesophageal specimens (33 biopsies and 17 resections) and 10 control specimens adjacent to tumour or normal oesophageal biopsies were studied. Specific antibodies were used to determine the cellular localisation of TK1, prokallikrein, and the kinin B1 and B2 receptors in normal and oesophageal specimens by standard immunohistochemical techniques. The intensity of immunolabelling was quantified by image analysis. Antisense probes for TK1 and the kinin B1 and B2 receptors were also used to localise mRNA. Results: TK1 (active and prokallikrein) was expressed in the mucosa of normal and tumour oesophageal epithelium. In general, expression was highest in activated mast cells, followed by giant tumour cells. Immunolabelling results were confirmed by ISH experiments. Conclusions: This is the first demonstration that TK1 and kinin B1 and B2 receptors are expressed in oesophageal carcinoma. Because TK1 released from tumour cells enzymatically generates mitogenic kinins from its endogenous substrate, kininogen, it is possible that third generation kinin receptor antagonists, which have been shown to be cytotoxic to cancer cells, may be useful therapeutic agents in this disease.