The effects of PTK787/ZK222584, an inhibitor of VEGFR and PDGFR? pathways, on intussusceptive angiogenesis and glomerular recovery from Thy1.1 nephritis

The aim of our study was to investigate the phenomenon of intussusceptive angiogenesis with a focus on its molecular regulation by vascular endothelial growth factor receptor (VEGFR)/platelet-derived growth factor receptor β (PDGFRβ) pathways and biological significance for glomerular recovery after acute injury. Glomerular healing by intussusception was examined in a particular setting of Thy1.1 nephritis, where the lysis of mesangial cells results in an initial collapse and successive rebuilding of glomerular capillary structure. Restoration of capillary structure after induction of Thy1.1 nephritis occurred by intussusceptive angiogenesis resulting in i) rapid expansion of the capillary plexus with reinstatement of the glomerular filtration surface and ii) restoration of the archetypical glomerular vascular pattern. Glomerular capillaries of nephritic rats after combined VEGFR2 and PDGFRβ inhibition by PTK787/ZK222584 (PTK/ZK) were tortuous and irregular. However, the onset of intussusceptive angiogenesis was influenced only after long-term PTK/ZK treatment, providing an important insight into differential molecular regulation between sprouting and intussusceptive angiogenesis. PTK/ZK treatment abolished α-smooth muscle actin and tensin expression by injured mesangial cells, impaired glomerular filtration of microspheres, and led to the reduction of glomerular volume and the presence of multiple hemorrhages detectable in the tubular system. Collectively, treatment of nephritic patients with PTK/ZK compound is not recommended.

Tight junctions in brain barriers during central nervous system inflammation

Homeostasis within the central nervous system (CNS) is a prerequisite to elicit proper neuronal function. The CNS is tightly sealed from the changeable milieu of the blood stream by the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier (BCSFB). Whereas the BBB is established by specialized endothelial cells of CNS microvessels, the BCSFB is formed by the epithelial cells of the choroid plexus. Both constitute physical barriers by a complex network of tight junctions (TJs) between adjacent cells. During many CNS inflammatory disorders, such as multiple sclerosis, human immunodeficiency virus infection, or Alzheimer's disease, production of pro-inflammatory cytokines, matrix metalloproteases, and reactive oxygen species are responsible for alterations of CNS barriers. Barrier dysfunction can contribute to neurological disorders in a passive way by vascular leakage of blood-borne molecules into the CNS and in an active way by guiding the migration of inflammatory cells into the CNS. Both ways may directly be linked to alterations in molecular composition, function, and dynamics of the TJ proteins. This review summarizes current knowledge on the cellular and molecular aspects of the functional and dysfunctional TJ complexes at the BBB and the BCSFB, with a particular emphasis on CNS inflammation and the role of reactive oxygen species.

The distribution of E-cadherin expression in listeric rhombencephalitis of ruminants indicates its involvement in Listeria monocytogenes neuroinvasion

AIM: To investigate the expression of E-cadherin, a major host cell receptor for Listeria monocytogenes (LM) internalin A, in the ruminant nervous system and its putative role in brainstem invasion and intracerebral spread of LM in the natural disease. METHODS: Immunohistochemistry and double immunofluorescence was performed on brains, cranial nerves and ganglia of ruminants with and without natural LM rhombencephalitis using antibodies against E-cadherin, protein gene product 9.5, myelin-associated glycoprotein and LM. RESULTS: In the ruminant brain, E-cadherin is expressed in choroid plexus epithelium, meningothelium and restricted neuropil areas of the medulla, but not in the endothelium. In cranial nerves and ganglia, E-cadherin is expressed in satellite cells and myelinating Schwann cells. Expression does not differ between ruminants with or without listeriosis and does not overlap with the presence of microabscesses in the medulla. LM is observed in phagocytes, axons, Schwann cells, satellite cells and ganglionic neurones. CONCLUSION: Our results support the view that the specific ligand-receptor interaction between LM and host E-cadherin is involved in the neuropathogenesis of ruminant listeriosis. They suggest that oral epithelium and Schwann cells expressing E-cadherin provide a port of entry for free bacteria offering a site of primary intracellular replication, from where the bacterium may invade the axonal compartment by cell-to-cell spread. As E-cadherin expression in the ruminant central nervous system is weak, only very locally restricted and not related to the presence of microabscesses, it is likely that further intracerebral spread is independent of E-cadherin and relies primarily on axonal spread.

GLP-2 receptors in human disease: high expression in gastrointestinal stromal tumors and Crohn's disease

Peptide hormones of the glucagon-like peptide (GLP) family play an increasing clinical role, as reported for GLP-1 in diabetes therapy and insulinoma diagnostics. GLP-2, despite its known trophic and anti-inflammatory intestinal actions translated into preliminary clinical studies using the GLP-2 analogue teduglutide for treatment of short bowel syndrome and Crohn's disease, remains poorly characterized in terms of expression of its receptor in tissues of interest. Therefore, the GLP-2 receptor expression was assessed in 237 tumor and 148 non-neoplastic tissue samples with in vitro receptor autoradiography. A GLP-2 receptor expression was present in 68% of gastrointestinal stromal tumors (GIST). Furthermore, GLP-2 receptors were identified in the intestinal myenteric plexus, with significant up-regulation in active Crohn's disease. The GLP-2 receptors in GIST may be used for clinical applications like in vivo targeting with radiolabelled GLP-2 analogues for imaging and therapy. Moreover, the over-expressed GLP-2 receptor in the myenteric plexus may represent the morphological correlate of the clinical target of teduglutide in Crohn's disease.

Angiotensinergic innervation of the kidney: Localization and relationship with catecholaminergic postganglionic and sensory nerve fibers

We describe an angiotensin (Ang) II-containing innervation of the kidney. Cryosections of rat, pig and human kidneys were investigated for the presence of Ang II-containing nerve fibers using a mouse monoclonal antibody against Ang II (4B3). Co-staining was performed with antibodies against synaptophysin, tyrosine 3-hydroxylase, and dopamine beta-hydroxylase to detect catecholaminergic efferent fibers and against calcitonin gene-related peptide to detect sensory fibers. Tagged secondary antibodies and confocal light or laser scanning microscopy were used for immunofluorescence detection. Ang II-containing nerve fibers were densely present in the renal pelvis, the subepithelial layer of the urothelium, the arterial nervous plexus, and the peritubular interstitium of the cortex and outer medulla. They were infrequent in central veins and the renal capsule and absent within glomeruli and the renal papilla. Ang II-positive fibers represented phenotypic subgroups of catecholaminergic postganglionic or sensory fibers with different morphology and intrarenal distribution compared to their Ang II-negative counterparts. The Ang II-positive postganglionic fibers were thicker, produced typically fusiform varicosities and preferentially innervated the outer medulla and periglomerular arterioles. Ang II-negative sensory fibers were highly varicose, prevailing in the pelvis and scarce in the renal periphery compared to the rarely varicose Ang II-positive fibers. Neurons within renal microganglia displayed angiotensinergic, catecholaminergic, or combined phenotypes. Our results suggest that autonomic fibers may be an independent source of intrarenal Ang II acting as a neuropeptide co-transmitter or neuromodulator. The angiotensinergic renal innervation may play a distinct role in the neuronal control of renal sodium reabsorption, vasomotion and renin secretion.

Junctional adhesion molecule (JAM)-C deficient C57BL/6 mice develop a severe hydrocephalus

The junctional adhesion molecule (JAM)-C is a widely expressed adhesion molecule regulating cell adhesion, cell polarity and inflammation. JAM-C expression and function in the central nervous system (CNS) has been poorly characterized to date. Here we show that JAM-C(-/-) mice backcrossed onto the C57BL/6 genetic background developed a severe hydrocephalus. An in depth immunohistochemical study revealed specific immunostaining for JAM-C in vascular endothelial cells in the CNS parenchyma, the meninges and in the choroid plexus of healthy C57BL/6 mice. Additional JAM-C immunostaining was detected on ependymal cells lining the ventricles and on choroid plexus epithelial cells. Despite the presence of hemorrhages in the brains of JAM-C(-/-) mice, our study demonstrates that development of the hydrocephalus was not due to a vascular function of JAM-C as endothelial re-expression of JAM-C failed to rescue the hydrocephalus phenotype of JAM-C(-/-) C57BL/6 mice. Evaluation of cerebrospinal fluid (CSF) circulation within the ventricular system of JAM-C(-/-) mice excluded occlusion of the cerebral aqueduct as the cause of hydrocephalus development but showed the acquisition of a block or reduction of CSF drainage from the lateral to the 3(rd) ventricle in JAM-C(-/-) C57BL/6 mice. Taken together, our study suggests that JAM-C(-/-) C57BL/6 mice model the important role for JAM-C in brain development and CSF homeostasis as recently observed in humans with a loss-of-function mutation in JAM-C.

Capture, crawl, cross: the T cell code to breach the blood-brain barriers

The central nervous system (CNS) is an immunologically privileged site to which access of circulating immune cells is tightly controlled by the endothelial blood-brain barrier (BBB; see Glossary) localized in CNS microvessels, and the epithelial blood-cerebrospinal fluid barrier (BCSFB) within the choroid plexus. As a result of the specialized structure of the CNS barriers, immune cell entry into the CNS parenchyma involves two differently regulated steps: migration of immune cells across the BBB or BCSFB into the cerebrospinal fluid (CSF)-drained spaces of the CNS, followed by progression across the glia limitans into the CNS parenchyma. With a focus on multiple sclerosis (MS) and its animal models, this review summarizes the distinct molecular mechanisms required for immune cell migration across the different CNS barriers.

T-cell trafficking in the central nervous system

To perform their distinct effector functions, pathogen-specific T cells have to migrate to target tissue where they recognize antigens and produce cytokines that elicit appropriate types of protective responses. Similarly, migration of pathogenic self-reactive T cells to target organs is an essential step required for tissue-specific autoimmunity. In this article, we review data from our laboratory as well as other laboratories that have established that effector function and migratory capacity are coordinately regulated in different T-cell subsets. We then describe how pathogenic T cells can enter into intact or inflamed central nervous system (CNS) to cause experimental autoimmune encephalomyelitis or multiple sclerosis. In particular, we elaborate on the role of CCR6/CCL20 axis in migration through the choroid plexus and the involvement of this pathway in immune surveillance of and autoimmunity in the CNS.

Regulation of immune cell entry into the central nervous system

The central nervous system (CNS) has long been regarded as an immune privileged organ implying that the immune system avoids the CNS to not disturb its homeostasis, which is critical for proper function of neurons. Meanwhile, it is accepted that immune cells do in fact gain access to the CNS and that immune responses can be mounted within this tissue. However, the unique CNS microenvironment strictly controls these immune reactions starting with tightly controlling immune cell entry into the tissue. The endothelial blood-brain barrier (BBB) and the epithelial blood-cerebrospinal fluid (CSF) barrier, which protect the CNS from the constantly changing milieu within the bloodstream, also strictly control immune cell entry into the CNS. Under physiological conditions, immune cell migration into the CNS is kept at a very low level. In contrast, during a variety of pathological conditions of the CNS such as viral or bacterial infections, or during inflammatory diseases such as multiple sclerosis, immunocompetent cells readily traverse the BBB and likely also the choroid plexus and subsequently enter the CNS parenchyma or CSF spaces. This chapter summarizes our current knowledge of immune cell entry across the blood CNS barriers. A large body of the currently available information on immune cell entry into the CNS has been derived from studying experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Therefore, most of this chapter discussing immune cell entry during CNS pathogenesis refers to observations in the EAE model, allowing for the possibility that other mechanisms of immune cell entry into the CNS might apply under different pathological conditions such as bacterial meningitis or stroke.

Distribution of muscarinic receptor subtypes and interstitial cells of Cajal in the gastrointestinal tract of healthy dairy cows

OBJECTIVE: To describe the distribution of muscarinic receptor subtypes M(1) to M(5) and interstitial cells of Cajal (ICCs) in the gastrointestinal tract of healthy dairy cows. SAMPLE POPULATION: Full-thickness samples were collected from the fundus, corpus, and pyloric part of the abomasum and from the duodenum, ileum, cecum, proximal loop of the ascending colon, and both external loops of the spiral colon of 5 healthy dairy cows after slaughter. PROCEDURES: Samples were fixed in paraformaldehyde and embedded in paraffin. Muscarinic receptor subtypes and ICCs were identified by immunohistochemical analysis. RESULTS: Staining for M(1) receptors was found in the submucosal plexus and myenteric plexus. Antibodies against M(2) receptors stained nuclei of smooth muscle cells only. Evidence of M(3) receptors was found in the lamina propria, in intramuscular neuronal terminals, on intermuscular nerve fibers, and on myocytes of microvessels. There was no staining for M(4) receptors. Staining for M(5) receptors was evident in the myocytes of microvessels and in smooth muscle cells. The ICCs were detected in the myenteric plexus and within smooth muscle layers. Distribution among locations of the bovine gastrointestinal tract did not differ for muscarinic receptor subtypes or ICCs. CONCLUSIONS AND CLINICAL RELEVANCE: The broad distribution of M(1), M(3), M(5), and ICCs in the bovine gastrointestinal tract indicated that these components are likely to play an important role in the regulation of gastrointestinal tract motility in healthy dairy cows. Muscarinic receptors and ICCs may be implicated in the pathogenesis of motility disorders, such as abomasal displacement and cecal dilatation-dislocation.

Increased proximal urethral sensory threshold after radical pelvic surgery in women

AIM: To identify factors that potentially influence urethral sensitivity in women. PATIENTS AND METHODS: The current perception threshold was measured by double ring electrodes in the proximal and distal urethra in 120 women. Univariate analysis using Kaplan-Meier models and multivariate analysis applying Cox regressions were performed to identify factors influencing urethral sensitivity in women. RESULTS: In univariate and multivariate analysis, women who had undergone radical pelvic surgery (radical cystectomy n = 12, radical rectal surgery n = 4) showed a significantly (log rank test P < 0.0001) increased proximal urethral sensory threshold compared to those without prior surgery (hazard ratio (HR) 4.17, 95% confidence interval (CI) 2.04-8.51), following vaginal hysterectomy (HR 4.95, 95% CI 2.07-11.85), abdominal hysterectomy (HR 5.96, 95% CI 2.68-13.23), or other non-pelvic surgery (HR 4.86, 95% CI 2.24-10.52). However, distal urethral sensitivity was unaffected by any form of prior surgery. Also other variables assessed, including age, concomitant diseases, urodynamic diagnoses, functional urethral length, and maximum urethral closure pressure at rest had no influence on urethral sensitivity in univariate as well as in multivariate analysis. CONCLUSIONS: Increased proximal but unaffected distal urethral sensory threshold after radical pelvic surgery in women suggests that the afferent nerve fibers from the proximal urethra mainly pass through the pelvic plexus which is prone to damage during radical pelvic surgery, whereas the afferent innervation of the distal urethra is provided by the pudendal nerve. Better understanding the innervation of the proximal and distal urethra may help to improve surgical procedures, especially nerve sparing techniques. Neurourol. Urodynam. (c) 2006 Wiley-Liss, Inc.

Nerve-sparing open radical retropubic prostatectomy

INTRODUCTION: In recent years, the surgical technique for open radical prostatectomy has evolved and increasing attention is paid to preserving anatomic structures and the impact on outcome and quality of life. METHODS: Technical aspects of nerve-sparing open radical retropubic prostatectomy (RRP) are described. Patient selection criteria and functional results are discussed, focusing on postoperative urinary continence. RESULTS: The video demonstrates the nerve-sparing open RRP and important steps are elucidated with schematic drawings. The value of nerve sparing, not only for preserving erectile function, but also for preserving urinary continence is discussed and results from our institution are presented. In our series, urinary incontinence was present in 1 of 71 patients (1%) with attempted bilateral nerve-sparing, 11 of 322 (3%) with attempted unilateral nerve-sparing, or 19 of 139 (14%) without attempted nerve-sparing surgery. In multiple logistic regression analysis, the only statistically significant factor influencing urinary continence after open RRP was attempted nerve sparing (odds ratio, 4.77; 95% confidence interval, 2.18-10.44; p=0.0001). CONCLUSIONS: Nerve-sparing surgery has a significant impact on erectile function and urinary continence and should be performed in all patients provided radical tumour resection is not compromised. For successful nerve preservation we advocate a lateral approach to the prostate to improve visualisation and simplify separation of the neurovascular bundles from the dorsolateral prostatic capsule. Bunching, ligating, and incising Santorini's plexus over the prostate and not over the sphincter ensures a bloodless surgical field. Mucosa-to-mucosa adaptation of the reconstructed bladder neck and the urethra is another important factor to be observed.

Microvascular endowment in the developing chicken embryo lung

In the current study, the contribution of the major angiogenic mechanisms, sprouting and intussusception, to vascular development in the avian lung has been demonstrated. Sprouting guides the emerging vessels to form the primordial vascular plexus, which successively surrounds and encloses the parabronchi. Intussusceptive angiogenesis has an upsurge from embryonic day 15 (E15) and contributes to the remarkably rapid expansion of the capillary plexus. Increased blood flow stimulates formation of pillars (the archetype of intussusception) in rows, their subsequent fusion and concomitant delineation of slender, solitary vascular entities from the disorganized meshwork, thus crafting the organ-specific angioarchitecture. Morphometric investigations revealed that sprouting is preponderant in the early period of development with a peak at E15 but is subsequently supplanted by intussusceptive angiogenesis by the time of hatching. Quantitative RT-PCR revealed that moderate levels of basic FGF (bFGF) and VEGF-A were maintained during the sprouting phase while PDGF-B remained minimal. All three factors were elevated during the intussusceptive phase. Immunohistoreactivity for VEGF was mainly in the epithelial cells, whereas bFGF was confined to the stromal compartment. Temporospatial interplay between sprouting and intussusceptive angiogenesis fabricates a unique vascular angioarchitecture that contributes to the establishment of a highly efficient gas exchange system characteristic of the avian lung.

Nitric oxide is protective in listeric meningoencephalitis of rats

The bacterium Listeria monocytogenes causes meningoencephalitis in humans. In rodents, listeriosis is associated with granulomatous lesions in the liver and the spleen, but not with meningoencephalitis. Here, infant rats were infected intracisternally to generate experimental listeric meningoencephalitis. Dose-dependent effects of intracisternal inoculation with L. monocytogenes on survival and activity were noted; 10(4) L. monocytogenes organisms induced a self-limiting brain infection. Bacteria invaded the basal meninges, chorioid plexus and ependyme, spread to subependymal tissue and hippocampus, and disappeared by day 7. This was paralleled by recruitment and subsequent disappearance of macrophages expressing inducible nitric oxide synthase (iNOS) and nitrotyrosine accumulation, an indication of nitric oxide (NO.) production. Treatment with the spin-trapping agent alpha-phenyl-tert-butyl nitrone (PBN) dramatically increased mortality and led to bacterial numbers in the brain 2 orders of magnitude higher than in control animals. Treatment with the selective iNOS inhibitor L-N(6)-(1-iminoethyl)-lysine (L-NIL) increased mortality to a similar extent and led to 1 order of magnitude higher bacterial counts in the brain, compared with controls. The numbers of bacteria that spread to the spleen and liver did not significantly differ among L-NIL-treated, PBN-treated, and control animals. Thus, the infant rat brain is able to mobilize powerful antilisterial mechanisms, and both reactive oxygen and NO. contribute to Listeria growth control.

Tumor recovery by angiogenic switch from sprouting to intussusceptive angiogenesis after treatment with PTK787/ZK222584 or ionizing radiation

Inhibitors of angiogenesis and radiation induce compensatory changes in the tumor vasculature both during and after treatment cessation. To assess the responses to irradiation and vascular endothelial growth factor-receptor tyrosine kinase inhibition (by the vascular endothelial growth factor tyrosine kinase inhibitor PTK787/ZK222854), mammary carcinoma allografts were investigated by vascular casting; electron, light, and confocal microscopy; and immunoblotting. Irradiation and anti-angiogenic therapy had similar effects on the tumor vasculature. Both treatments reduced tumor vascularization, particularly in the tumor medulla. After cessation of therapy, the tumor vasculature expanded predominantly by intussusception with a plexus composed of enlarged sinusoidal-like vessels containing multiple transluminal tissue pillars. Tumor revascularization originated from preserved alpha-smooth muscle actin-positive vessels in the tumor cortex. Quantification revealed that recovery was characterized by an angiogenic switch from sprouting to intussusception. Up-regulated alpha-smooth muscle actin-expression during recovery reflected the recruitment of alpha-smooth muscle actin-positive cells for intussusception as part of the angio-adaptive mechanism. Tumor recovery was associated with a dramatic decrease (by 30% to 40%) in the intratumoral microvascular density, probably as a result of intussusceptive pruning and, surprisingly, with only a minimal reduction of the total microvascular (exchange) area. Therefore, the vascular supply to the tumor was not severely compromised, as demonstrated by hypoxia-inducible factor-1alpha expression. Both irradiation and anti-angiogenic therapy cause a switch from sprouting to intussusceptive angiogenesis, representing an escape mechanism and accounting for the development of resistance, as well as rapid recovery, after cessation of therapy.