Andrea Lippoldt

Tight junctions of the blood–brain barrier: development, composition and regulation

1. The blood-brain barrier is essential for the maintenance and regulation of the neural microenvironment. The main characteristic features of blood-brain barrier endothelial cells are an extremely low rate of transcytotic vesicles and a restrictive paracellular diffusion barrier. 2. Endothelial blood-brain barrier tight junctions differ from epithelial tight junctions, not only by distinct morphological and molecular properties, but also by the fact that endothelial tight junctions are more sensitive to microenvironmental than epithelial factors. 3. Many ubiquitous molecular tight junction components have been identified and characterized including claudins, occludin, ZO-1, ZO-2, ZO-3, cingulin and 7H6. Signaling pathways involved in tight junction regulation include G-proteins, serine-, threonine- and tyrosine-kinases, extra and intracellular calcium levels, cAMP levels, proteases and cytokines. Common to most of these pathways is the modulation of cytoskeletal elements and the connection of tight junction transmembrane molecules to the cytoskeleton. Additionally, crosstalk between components of the tight junction- and the cadherin-catenin system of the adherens junction suggests a close functional interdependence of the two cell-cell contact systems. 4. Important new molecular aspects of tight junction regulation were recently elucidated. This review provides an integration of these new results.

Hypertension-Induced End-Organ Damage: A New Transgenic Approach to an Old Problem

Angiotensin (Ang) II-induced organ damage has fascinated students of hypertension since the work of Wilson and Byrom. We are investigating a double transgenic rat (dTGR) model, in which rats transgenic for the human angiotensinogen and renin genes are crossed. These rats develop moderately severe hypertension but die of end-organ cardiac and renal damage by week 7. The heart shows necrosis and fibrosis, whereas the kidneys resemble the hemolytic-uremic syndrome vasculopathy. Surface adhesion molecules (ICAM-1 and VCAM-1) are expressed early on the endothelium, while the corresponding ligands are found on circulating leukocytes. Leukocyte infiltration in the vascular wall accompanies PAI-1, MCP-1, and VEGF expression. The expression of TGF-beta and deposition of extracellular matrix proteins follows, which is accompanied by fibrinoid vasculitis in small vessels of the heart and kidneys. Angiotensin-converting enzyme inhibitors and AT1 receptor blockers each lowered blood pressure and shifted pressure natriuresis partially leftward by different mechanisms. When combined, they normalized blood pressure, pressure natriuresis, and protected from vasculopathy completely. Renin inhibition lowered blood pressure partially, but protected from vasculopathy completely. Endothelin receptor blockade had no influence on blood pressure but protected from vasculopathy and improved survival. We show evidence that Ang II stimulates oxidative stress directly or indirectly via endothelin 1 and that NFkappaB is upregulated in this model. We speculate that the transcription factors NFkappaB and AP-1 are involved with initiating chemokine and cytokine expression, leading to the above cascade. The unique model and our pharmacological probes will enable us to test these hypotheses.

High Human Renin Hypertension in Transgenic Rats

We developed a model of spontaneously high human renin hypertension in the rat by producing two transgenic strains, one for human angiotensinogen with the endogenous promoter and one for human renin with the endogenous promoter. Neither transgenic strain was hypertensive. These strains were then crossed, producing a double transgenic strain. The double transgenic rats, both males and females, developed severe hypertension (mean systolic pressure, 200 mm Hg) and died after a mean of 55 days if untreated. The rats had a human plasma renin concentration of 269 +/- 381 (+/-SD) ng angiotensin I (Ang I)/mL per hour, plasma renin activity of 177 +/- 176 ng Ang I/mL per hour, rat angiotensinogen concentration of 1.49 +/- 1 microgram Ang I/mL, and human angiotensinogen concentration of 78 +/- 39 micrograms Ang I/mL (n = 49). Control rats had plasma renin activity of 3.7 +/- 3.9 ng Ang I/mL per hour and rat angiotensinogen of 1.32 +/- 0.16 micrograms Ang I/mL. Angiotensinogen transgene expression by RNase protection assay was ubiquitously present but most prominent in liver. Renin transgene expression was high in kidney but absent in liver. The rats featured severe cardiac hypertrophy, with increased cross section of cardiomyocytes but little myocardial fibrosis. The kidneys showed atrophic tubules, thickened vessel walls, and increased interstitium. Both the angiotensin-converting enzyme inhibitor lisinopril and the specific human renin inhibitor remikiren lowered blood pressure to normal values. Double transgenic mice have been developed that exhibit features quite similar to those described here; their gene expressions are similar. The specificity of rodent and human renin is similarly documented. Although many elegant physiological studies can now be done in mice, rats nevertheless offer flexibility, particularly in terms of detailed cardiac and renal physiology and pharmacology. We conclude that this double transgenic strain will facilitate simultaneous investigation of genetic and pathophysiological aspects of renin-induced hypertension. The fact that human renin can be studied in the rat is a unique feature of this model.

Structural alterations of tight junctions are associated with loss of polarity in stroke-prone spontaneously hypertensive rat blood-brain barrier endothelial cells.

The mechanisms leading to stroke in stroke-prone spontaneously hypertensive rats (SHRSP) are not well understood. We tested the hypothesis that the endothelial tight junctions of the blood-brain barrier are altered in SHRSP prior to stroke. We investigated tight junctions in 13-week-old SHRSP, spontaneously hypertensive stroke-resistant rats (SHR) and age-matched Wistar-Kyoto rats (WKY) by electron microscopy and immunocytochemistry. Ultrathin sections showed no difference in junction structure of cerebral capillaries from SHRSP, SHR and WKY, respectively. However, using freeze-fracturing, we observed that the blood-brain barrier specific distribution of tight junction particles between P- and E-face in WKY (58.7+/-3.6%, P-face; 41.2+/-5.59%, E-face) and SHR (53.2+/-19. 3%, P-face; 55.6+/-13.25%, E-face) was changed to an 89.4+/-9.9% predominant E-face association in cerebral capillaries from SHRSP. However, the expression of the tight junction molecules ZO-1, occludin, claudin-1 and claudin-5 was not changed in capillaries of SHRSP. Permeability of brain capillaries from SHRSP was not different compared to SHR and WKY using lanthanum nitrate as a tracer. In contrast, analysis of endothelial cell polarity by distribution of the glucose-1 transporter (Glut-1) revealed that its abluminal:luminal ratio was reduced from 4:1 in SHR and WKY to 1:1 in endothelial cells of cerebral capillaries of SHRSP. In summary, we demonstrate that early changes exist in cerebral capillaries from a genetic model of hypertension-associated stroke. We suggest that a disturbed fence function of the tight junctions in SHRSP blood-brain barrier endothelial cells may lead to subtle changes in polarity. These changes may contribute to the pathogenesis of stroke.

Organization of choroid plexus epithelial and endothelial cell tight junctions and regulation of claudin-1, -2 and -5 expression by protein kinase C.

&NA; Claudins are components of the tight junctional complex in epithelial and endothelial cells. We characterized the composition of tight junctions in the choroid plexus of the lateral ventricle in the rat brain and tested whether protein kinase C induced changes in their composition. Claudin‐1, −2 and −5 were present in the epithelial cells at and near the tight junctions, respectively. In the endothelial cells, claudin‐5 was stronger expressed than claudin‐1 and ‐2. Twenty‐four hours after the phorbolester injection into the ventricle, claudin‐1 immunoreactivity of the epithelial cells was increased and spread to the cytoplasm. The claudin‐2 and ‐5 immunoreactivities were reduced. These findings are consistent with an influence of protein kinase C on the composition of the tight junctions in the choroid plexus.

Renovascular Hypertension in Bradykinin B2-Receptor Knockout Mice

We evaluated whether kinins exert a protective action against the development of two-kidney, one clip (2K1C) hypertension, a model characterized by an activated renin-angiotensin system in the ischemic kidney and increased expression of the bradykinin (BK) B2 receptor in the contralateral kidney. BK B2-receptor knockout (B2-/-), wild-type (B2+/+), and heterozygous (B2+/-) mice underwent clipping of the left renal artery, with the other kidney remaining untouched. Basal systolic blood pressure (SBP, via tail-cuff plethysmography) was higher in B2-/- mice than in B2+/- or B2+/+ mice (121+/-2 versus 113+/-2 and 109+/-1 mm Hg; P<0.05 for both comparisons). SBP did not change from basal values after sham operation, but it increased in mice that underwent clipping. The increase in SBP was greater in 2K1C B2-/- mice than in B2+/- or B2+/+ mice (28+/-2 versus 14+/-2 and 14+/-2 mm Hg, respectively, at 2 weeks; P<0.05 for both comparisons). Blockade of the BK B2 receptor by Icatibant enhanced the pressure response to clipping in B2+/+ mice (29+/-2 mm Hg at 2 weeks). Intra-arterial mean blood pressure (MBP) was higher in 2K1C than in respective sham-operated mice, with the MBP difference being higher in B2-/- mice (32 and 38 mm Hg, at 2 and 4 weeks, respectively), and higher in B2+/+ mice given Icatibant (30 and 32 mm Hg) than in B2+/+ mice without Icatibant (17 and 18 mm Hg). At 4 weeks, acute injection of an angiotensin type 1 receptor antagonist normalized the MBP of 2K1C hypertensive mice. A tachycardic response was observed 1 week after clipping in B2-/- and B2+/- mice, but this effect was delayed in B2+/+ mice. However, the HR response to clipping in B2+/+ mice was enhanced by Icatibant. Within each strain, heart weight to body weight ratio was greater in 2K1C hypertensive mice than in sham-operated control animals (B2-/-: 5.7+/-0.1 versus 5.2+/-0.1; B2+/+: 5.1+/-0.1 versus 4.5+/-0.1; P<0.01 for both comparisons). The clipped kidney weight to nonclipped kidney weight ratio was consistently reduced in mice with 2K1C hypertension. Our results indicate that kinins acting on the BK B2 receptor exert a protective action against excessive blood pressure elevation during early phases of 2K1C hypertension.

Altered gene expression in cerebral capillaries of stroke-prone spontaneously hypertensive rats

Stroke-prone spontaneously hypertensive rats (SHRSP) are a well-characterized, genetic model for stroke. We showed earlier that the structure and function of the tight junctions in SHRSP blood-brain barrier endothelial cells is disturbed prior to stroke. To investigate the molecular events leading to endothelial dysfunction in SHRSP cerebral capillaries, we carried out suppression subtractive hybridization (SSH) in combination with a cDNA filter screening step. We identified two cDNA fragments that were upregulated in SHRSP, compared to stroke-resistant spontaneously hypertensive rats (SHR), and found open reading frames of 133 and 138 amino acids, respectively. These peptides did not match any known proteins in public databases. A third upregulated SHRSP cDNA fragment was identified as the rat sulfonylurea receptor 2B (SUR2B). We also isolated and cloned the cDNA of the rat homologue for the mouse G-protein signaling 5 (RGS5) regulator. This regulator was downregulated in SHRSP. We used in situ hybridization to show that rat RGS5 is expressed in the brain capillary endothelium and in the choroid plexus. Our findings may lead to the identification of new stroke-related genes.

The Brain Renin-Angiotensin System: Molecular Mechanisms of Cell to Cell Interactions

The components of the Renin-Angiotensin System (RAS) have been found to be expressed in the brain. Angiotensinogen, the high molecular weight precursor of the system, is widely distributed and expressed in areas not related to control of blood pressure and body fluid homeostasis as well. It has been shown that it is regulated by steroid hormones independently from the liver and that it is also regulated in a different manner in several brain areas. Angiotensin II, the effector peptide of the system, may be generated in the brain via the classical pathway, using renin and angiotensin converting enzyme or directly from angiotensinogen by cathepsin G or tonin. N-terminal peptides of angiotensin II have been found in several brain areas with ANG (1-7) involved in vasopressin release however without influence on blood pressure and with ANG III acting as potent as ANG II. Transgenic animals may be used to study the pathophysiology of an activated brain RAS.

Effect of Captopril and Angiotensin II Receptor Blockade on Pressure Natriuresis in Transgenic TGR(mRen-2)27 Rats

The pressure-natriuresis curve of transgenic rats harboring an extra mouse renin gene [TGR(mRen-2)27] is shifted rightward compared with controls; however, whether intrarenal angiotensin II effects are responsible for the rightward shift is unknown. To clarify this issue we infused the converting enzyme inhibitor captopril or the angiotensin II receptor blocker CV 11974 into transgenic and normotensive Sprague-Dawley Hannover control rats. We eliminated any other neural or endocrine regulatory differences between transgenic and control rats by renal denervation and infusion of vasopressin, aldosterone, corticosterone, and norepinephrine in sufficient quantities to occupy all receptors. Sodium excretion increased from 3.4 +/- 1.2 to 10.1 +/- 0.5 mumol/min per gram kidney weight in transgenic rats when renal perfusion pressure was increased from 158 to 201 mm Hg. Captopril (4 mg/kg) and CV 11974 (0.1 mg/kg) shifted the pressure-natriuresis curve of transgenic rats leftward, so that sodium excretion was threefold higher at similar renal perfusion pressures (150 to 160 mm Hg). Similarly, fractional sodium and water excretion curves were shifted leftward, so that values for transgenic and control rats were no longer different. Over the pressure range, renal blood flow in transgenic rats ranged from 3.1 +/- 0.7 to 4.4 +/- 0.5 mL/min per gram kidney weight and increased (P < .05) with both captopril and CV 11974 to ranges from 4.8 +/- 0.9 to 6.8 +/- 0.6 or from 4.5 +/- 0.7 to 6.9 +/- 1.0 mL/min per gram kidney weight, respectively. Glomerular filtration rate in transgenic rats, on the other hand, was not increased. Transgenic kidneys showed severe hypertension-induced nephrosclerosis.(ABSTRACT TRUNCATED AT 250 WORDS)

High levels of human chymase expression in the pineal and pituitary glands

The brain renin-angiotensin system plays a role in both cardiovascular homeostasis and neurosecretory functions. Since the mechanisms of angiotensin (Ang) II formation in the human brain have not been clarified, the aims of the present study were to determine the presence of human chymase and angiotensin I-converting enzyme (ACE) in human and non-human brains. In the human brain, the total Ang II-forming activity was significantly higher in the pineal and pituitary glands than those in other regions. In other species (rat, bovine and porcine), the level of chymase as well as total Ang II-forming activities in pineal glands were significantly lower than those in human glands. High levels of chymase-like immunoreactivity (ir) were found in the arteriolar endothelial cells, adventitial mesenchymal cells and in parenchymal cells of the human pineal and pituitary glands while ACE-ir was mostly observed in the endothelial cells and occasionally found in parenchymal cells. Our study provides the first evidence that human chymase exists in the pineal and pituitary glands. The remarkable regional and species differences in mechanisms of Ang II formation suggest a specific role of chymase or ACE in the human brain.