Faculty Bibliography

BACKGROUND: Spontaneous intracranial hypotension is an uncommon clinical entity. Heritable connective tissue disorders (HCTD), such as Marfan syndrome, are frequently implicated as an underlying cause, due to dural structural weaknesses that predispose patients to spontaneous cerebrospinal fluid (CSF) leak. Due to the high prevalence of multi-system disease in HCTD, diagnosis and treatment are often complicated. CASE DESCRIPTION: We present a 58-year-old female with Marfan syndrome on anticoagulation for a mechanical aortic valve replacement who came to medical attention with severe, acute-onset headache following a straining episode. Noninvasive magnetic resonance (MR) myelography confirmed thoracic CSF extravasations and multiple lumbar diverticula. The patient was treated conservatively and her symptoms resolved. CONCLUSION: We discuss the common presentation, diagnostic tools, and treatment options for spontaneous CSF leaks in patients with Marfan syndrome or related HCTD with an emphasis on noninvasive modalities and a review of the major radiographic criteria used to diagnose dural abnormalities, such as dural ectasia.

Byproducts of cytokine activation are sometimes useful as surrogate biomarkers for monitoring cytokine generation in patients. Transforming growth factor (TGF)-beta plays a pivotal role in pathogenesis of hepatic fibrosis. TGF-beta is produced as part of an inactive latent complex, in which the cytokine is trapped by its propeptide, the latency-associated protein (LAP). Therefore, to exert its biological activity, TGF-beta must be released from the latent complex. Several proteases activate latent TGF-beta by cutting LAP. We previously reported that Camostat Mesilate, a broad spectrum protease inhibitor, which is especially potent at inhibiting plasma kallikrein (PLK), prevented liver fibrosis in the porcine serum-induced liver fibrosis model in rats. We suggested that PLK may work as an activator of latent TGF-beta during the pathogenesis of liver diseases in the animal models. However, it remained to be elucidated whether this activation mechanism also functions in fibrotic liver in patients. Here, we report that PLK cleaves LAP between R(58) and L(59) residues. We have produced monoclonal antibodies against two degradation products of LAP (LAP-DP) by PLK, and we have used these specific antibodies to immunostain LAP-DP in liver tissues from both fibrotic animals and patients. The N-terminal side LAP-DP ending at R(58) (R(58) LAP-DP) was detected in liver tissues, while the C-terminal side LAP-DP beginning at L(59) (L(59) LAP-DP) was not detectable. The R(58) LAP-DP was seen mostly in alpha-smooth muscle actin-positive activated stellate cells. These data suggest for the first time that the occurrence of a PLK-dependent TGF-beta activation reaction in patients and indicates that the LAP-DP may be useful as a surrogate marker reflecting PLK-dependent TGF-beta activation in fibrotic liver both in animal models and in patients.

Dopamine and estradiol interact in the regulation of lactotroph cell proliferation and prolactin secretion. Ablation of the dopamine D2 receptor gene (Drd2(-/-)) in mice leads to a sexually dimorphic phenotype of hyperprolactinemia and pituitary hyperplasia, which is stronger in females. TGF-beta1 is a known inhibitor of lactotroph proliferation. TGF-beta1 is regulated by dopamine and estradiol, and it is usually down-regulated in prolactinoma experimental models. To understand the role of TGF-beta1 in the gender-specific development of prolactinomas in Drd2(-/-) mice, we compared the expression of different components of the pituitary TGF-beta1 system, including active cytokine content, latent TGF-beta-binding protein isoforms, and possible local TGF-beta1 activators, in males and females in this model. Furthermore, we evaluated the effects of dopamine and estradiol administration to elucidate their role in TGF-beta1 system regulation. The expression of active TGF-beta1, latent TGF-beta-binding protein isoforms, and several putative TGF-beta1 activators evaluated was higher in male than in female mouse pituitary glands. However, Drd2(-/-) female mice were more sensitive to the decrease in active TGF-beta1 content, as reflected by the down-regulation of TGF-beta1 target genes. Estrogen and dopamine caused differential regulation of several components of the TGF-beta1 system. In particular, we found sex- and genotype- dependent regulation of active TGF-beta1 content and a similar expression pattern for 2 of the putative TGF-beta1 activators, thrombospondin-1 and kallikrein-1, suggesting that these proteins could mediate TGF-beta1 activation elicited by dopamine and estradiol. Our results indicate that (1) the loss of dopaminergic tone affects the pituitary TGF-beta1 system more strongly in females than in males, (2) males express higher levels of pituitary TGF-beta1 system components including active cytokine, and (3) estradiol negatively controls most of the components of the system. Because TGF-beta1 inhibits lactotroph proliferation, we propose that the higher levels of the TGF-beta1 system in males could protect or delay the development of prolactinomas in Drd2(-/-) male mice.

TGFbeta is secreted in a latent state and must be "activated" by molecules that facilitate its release from a latent complex and allow binding to high affinity cell surface receptors. Numerous molecules have been implicated as potential mediators of this activation process, but only a limited number of these activators have been demonstrated to play a role in TGFbeta mobilisation in vivo. Here we review the process of TGFbeta secretion and activation using evolutionary data, sequence conservation and structural information to examine the molecular mechanisms by which TGFbeta is secreted, sequestered and released. This allows the separation of more ancient TGFbeta activators from those factors that emerged more recently, and helps to define a potential hierarchy of activation mechanisms.

Lymphocyte homing, which contributes to inflammation, has been studied extensively in the small intestine, but there is little known about homing to the large intestine, the site most commonly affected in inflammatory bowel disease. GPR15, an orphan G protein-coupled receptor, controlled the specific homing of T cells, particularly FOXP3+ regulatory T cells (Tregs), to the large intestine lamina propria (LILP). GPR15 expression was modulated by gut microbiota and transforming growth factor-beta1, but not by retinoic acid. GPR15-deficient mice were prone to develop more severe large intestine inflammation, which was rescued by the transfer of GPR15-sufficient Tregs. Our findings thus describe a T cell homing receptor for LILP and indicate that GPR15 plays a role in mucosal immune tolerance largely by regulating the influx of Tregs.

Elastic fiber assembly requires deposition of elastin monomers onto microfibrils, the mechanism of which is incompletely understood. Here we show that latent TGF-beta binding protein 4 (LTBP-4) potentiates formation of elastic fibers through interacting with fibulin-5, a tropoelastin-binding protein necessary for elastogenesis. Decreased expression of LTBP-4 in human dermal fibroblast cells by siRNA treatment abolished the linear deposition of fibulin-5 and tropoelastin on microfibrils. It is notable that the addition of recombinant LTBP-4 to cell culture medium promoted elastin deposition on microfibrils without changing the expression of elastic fiber components. This elastogenic property of LTBP-4 is independent of bound TGF-beta because TGF-beta-free recombinant LTBP-4 was as potent an elastogenic inducer as TGF-beta-bound recombinant LTBP-4. Without LTBP-4, fibulin-5 and tropoelastin deposition was discontinuous and punctate in vitro and in vivo. These data suggest a unique function for LTBP-4 during elastic fibrogenesis, making it a potential therapeutic target for elastic fiber regeneration.

TGF-beta and its signaling pathways are important mediators in the suppression of cancers of the gastrointestinal tract. TGF-beta is released from cells in a latent complex consisting of TGF-beta, the TGF-beta propeptide [latency associated protein (LAP)], and a latent TGF-beta binding protein (LTBP). We previously generated mice in which the LTBP-binding cysteine residues in LAP TGF-beta1 were mutated to serine precluding covalent interactions with LTBP. These Tgfb1(C33S/C33S) mice develop multiorgan inflammation and tumors consistent with reduced TGF-beta1 activity. To test whether further reduction in active TGF-beta levels would yield additional tumors and a phenotype more similar to Tgfb1(-/-) mice, we generated mice that express TGF-beta1(C33S) and are deficient in either integrin beta8 or TSP-1, known activators of latent TGF-beta1. In addition, we generated mice that have one mutant allele and one null allele at the Tgfb1 locus, reasoning that these mice should synthesize half the total amount of TGF-beta1 as Tgfb1(C33S/C33S) mice, and the amount of active TGF-beta1 would be correspondingly decreased compared with Tgfb1(C33S/C33S) mice. These compound-mutant mice displayed more severe inflammation and higher tumor numbers than the parental Tgfb1(C33S/C33S) animals. The level of active TGF-beta1 in compound mutant mice seemed to be decreased compared with Tgfb1(C33S/C33S) mice as determined from analyses of surrogate markers of active TGF-beta, such as P-Smad2, C-Myc, KI-67, and markers of cell-cycle traverse. We conclude that these mutant mice provide a useful system for modulating TGF-beta levels in a manner that determines tumor number and inflammation within the gastrointestinal tract. Cancer Res; 73(1); 459-68. (c)2012 AACR.

Fibrillin microfibrils are extracellular matrix structures with essential functions in the development and the organization of tissues including blood vessels, bone, limbs and the eye. Fibrillin-1 and fibrillin-2 form the core of fibrillin microfibrils, to which multiple proteins associate to form a highly organized structure. Defining the components of this structure and their interactions is crucial to understand the pathobiology of microfibrillopathies associated with mutations in fibrillins and in microfibril-associated molecules. In this study, we have analyzed both in vitro and in vivo the role of fibrillin microfibrils in the matrix deposition of latent TGF-beta binding protein 1 (LTBP-1), -3 and -4; the three LTBPs that form a complex with TGF-beta. In Fbn1(-/-) ascending aortas and lungs, LTBP-3 and LTBP-4 are not incorporated into a matrix lacking fibrillin-1 microfibrils, whereas LTBP-1 is still deposited. In addition, in cultures of Fbn1(-/-) smooth muscle cells or lung fibroblasts, LTBP-3 and LTBP-4 are not incorporated into a matrix lacking fibrillin-1 microfibrils, whereas LTBP-1 is still deposited. Fibrillin-2 is not involved in the deposition of LTBP-1 in Fbn1(-/-) extracellular matrix as cells deficient for both fibrillin-1 and fibrillin-2 still incorporate LTBP-1 in their matrix. However, blocking the formation of the fibronectin network in Fbn1(-/-) cells abrogates the deposition of LTBP-1. Together, these data indicate that LTBP-3 and LTBP-4 association with the matrix depends on fibrillin-1 microfibrils, whereas LTBP-1 association depends on a fibronectin network. J. Cell. Physiol. 227: 3828-3836, 2012. (c) 2012 Wiley Periodicals, Inc.

The cytokine transforming growth factor-beta (TGF-beta) has multiple effects in both physiological and pathological conditions. TGF-beta is secreted as part of a tripartite complex from which it must be released in order to bind to its receptor. Sequestration of latent TGF-beta in the extracellular matrix (ECM) is crucial for proper mobilization of the latent cytokine and its activation. However, contrary to expectation, loss-of-function mutations in genes encoding certain matrix proteins that bind TGF-beta yield elevated, rather than decreased, TGF-beta levels, posing a 'TGF-beta paradox.' In this review, we discuss recent findings concerning the relationship of TGF-beta, ECM molecules, and latent TGF-beta activation and propose a model to resolve the 'TGF-beta paradox.'

Prolactinomas are the most prevalent type of secreting pituitary tumors in humans and generally respond well to a medical therapy with dopamine agonists. However, for patients exhibiting resistance to dopaminergic drugs, alternative treatments are desired. Antiangiogenic strategies might represent a potential therapy for these tumors. Thrombospondin 1 (TSP-1) is a large multifunctional glycoprotein involved in multiple biological processes including angiogenesis, apoptosis, and activation of TGF-beta1. Because tumors that overexpress TSP-1 grow more slowly, have fewer metastases, and have decreased angiogenesis, TSP-1 provides a novel target for cancer treatment. ABT-510 and ABT-898 are TSP-1 synthetic analogs that mimic its antiangiogenic action. In the present study, we explored the potential effect of ABT-510 and ABT-898 on experimental prolactinomas induced by chronic diethylstilbestrol (DES) treatment in female rats. We demonstrated that a 2-wk treatment with ABT-510 and ABT-898 counteracted the increase in pituitary size and serum prolactin levels as well as the pituitary proliferation rate induced by DES. These inhibitory effects on tumor growth could be mediated by the antiangiogenic properties of the drugs. We also demonstrated that ABT-510 and ABT-898, in addition to their described antiangiogenic effects, increased active TGF-beta1 level in the tumors. We postulate that the recovery of the local cytokine activation participates in the inhibition of lactotrope function. These results place these synthetic TSP-1 analogs as potential alternative or complementary treatments in dopamine agonist-resistant prolactinomas.