Joon W. Shim

Inactivation of Lrp5 in osteocytes reduces Young's modulus and responsiveness to the mechanical loading

Low-density-lipoprotein receptor-related protein 5 (Lrp5) is a co-receptor in Wnt signaling, which plays a critical role in development and maintenance of bone. Osteoporosis-pseudoglioma syndrome, for instance, arises from loss-of-function mutations in Lrp5, and global deletion of Lrp5 in mice results in significantly lower bone mineral density. Since osteocytes are proposed to act as a mechanosensor in the bone, we addressed a question whether a conditional loss-of-function mutation of Lrp5 selective to osteocytes (Dmp1-Cre;Lrp5(f/f)) would alter responses to ulna loading. Loading was applied to the right ulna for 3 min (360 cycles at 2Hz) at a peak force of 2.65 N for 3 consecutive days, and the contralateral ulna was used as a non-loaded control. Youngs modulus was determined using a midshaft section of the femur. The results showed that compared to age-matched littermate controls, mice lacking Lrp5 in osteocytes exhibited smaller skeletal size with reduced bone mineral density and content. Compared to controls, Lrp5 deletion in osteocytes also led to a 4.6-fold reduction in Youngs modulus. In response to ulna loading, mineralizing surface, mineral apposition rate, and bone formation rate were diminished in mice lacking Lrp5 in osteocytes by 52%, 85%, and 69%, respectively. Collectively, the results support the notion that the loss-of-function mutation of Lrp5 in osteocytes causes suppression of mechanoresponsiveness and reduces bone mass and Youngs modulus. In summary, Lrp5-mediated Wnt signaling significantly contributes to maintenance of mechanical properties and bone mass.

VEGF, which is elevated in the CSF of patients with hydrocephalus, causes ventriculomegaly and ependymal changes in rats

Hydrocephalus is a condition characterized primarily by excessive accumulation of fluid in the ventricles of the brain for which there is currently no effective pharmacological treatment. Surgery, often accompanied by complications, is the only current treatment. Extensive research in our laboratory along with work from others has suggested a link between hydrocephalus and vascular function. We hypothesized that vascular endothelial growth factor (VEGF), the major angiogenic factor, could play a role in the pathogenesis of hydrocephalus. We tested this hypothesis by examining two predictions of such a link: first, that VEGF is present in many cases of clinical hydrocephalus; and second, that exogenous VEGF in an animal model could cause ventricular enlargement and tissue changes associated with hydrocephalus. Our results support the idea that VEGF elevation can potentiate hydrocephalus. The clinical relevance of this work is that anti-angiogenic drugs may be useful in patients with hydrocephalus, either alone or in combination with the currently available surgical treatments.

Construction of a Tissue‐Engineered Annulus Fibrosus

The intervertebral disc is composed of load-bearing fibrocartilage that may be subjected to compressive forces up to 10 times the body weight. The multilaminated outer layer, the annulus fibrosus (AF), is vulnerable to damage and its regenerative potential is limited, sometimes leading to nuclear herniation. Scaffold-based tissue engineering of AF using stem cell technology has enabled the development of bi-laminate constructs after 10 weeks of culture. It is difficult to know if these constructs are limited by the differentiation state of the stem cells or the culture system. In this study, we have characterized an expandable scaffold-free neoconstruct using autologous AF cells. The construct was prepared from pellet cultures derived from monolayer cultures of AF cells from mature pigs that became embedded in their own extracellular matrix. The pellet cultures were incubated for 24 h in a standardized conical tube and then carefully transferred intact to a culture flask and incubated for 21 days to allow continued matrix synthesis. Cell viability was maintained above 90% throughout the culture period. The engineered scaffold-free construct was compared with the native AF tissue by characterization of gene expression of representative markers, histological architecture, and biochemical composition. The morphological and biochemical characteristics of the cultured disc construct are very similar to that of native AF. The cell number per gram of construct was equal to that of native AF. Expression of aggrecan was elevated in the engineered construct compared with RNA extracted from the AF. The glycosaminoglycan content in the engineered construct showed no significant difference to that from native construct. These data indicate that scaffold-free tissue constructs prepared from AF cells using a pellet-culture format may be useful for in vitro expansion for transplantation into damaged discs.

VEGF-A is elevated in CSF of pediatric patients undergoing surgery for hydrocephalus

Background Vascular endothelial growth factor A (VEGF-A) is a member of the larger family of VEGF-related cytokines that mediates multiple functions of endothelial cells including proliferation, migration, and permeability. Current thinking on the pathogenesis of communicating hydrocephalus focuses on pulsatile vascular mechanisms, but the biochemical and biological underpinnings remain obscure. Insertion of a shunt, the most common treatment of this disorder, often fails. We wished to begin exploration of a link between hyperpulsatility and increased VEGF, which is a potent vascular permeability factor. The success of anti-VEGF treatments in very low dose for edema associated with macular degeneration and diabetic retinopathy suggest that anti-VEGF treatment in hydrocephalus could in some cases be a therapeutic alternative to shunt insertion.

VEGF Signaling in Neurological Disorders

Vascular endothelial growth factor (VEGF) is a potent growth factor playing diverse roles in vasculogenesis and angiogenesis. In the brain, VEGF mediates angiogenesis, neural migration and neuroprotection. As a permeability factor, excessive VEGF disrupts intracellular barriers, increases leakage of the choroid plexus endothelia, evokes edema, and activates the inflammatory pathway. Recently, we discovered that a heparin binding epidermal growth factor like growth factor (HB-EGF)—a class of EGF receptor (EGFR) family ligands—contributes to the development of hydrocephalus with subarachnoid hemorrhage through activation of VEGF signaling. The objective of this review is to entail a recent update on causes of death due to neurological disorders involving cerebrovascular and age-related neurological conditions and to understand the mechanism by which angiogenesis-dependent pathological events can be treated with VEGF antagonisms. The Global Burden of Disease study indicates that cancer and cardiovascular disease including ischemic and hemorrhagic stroke are two leading causes of death worldwide. The literature suggests that VEGF signaling in ischemic brains highlights the importance of concentration, timing, and alternate route of modulating VEGF signaling pathway. Molecular targets distinguishing two distinct pathways of VEGF signaling may provide novel therapies for the treatment of neurological disorders and for maintaining lower mortality due to these conditions.

Excess HB-EGF, which promotes VEGF signaling, leads to hydrocephalus.

Heparin binding epidermal growth factor-like growth factor (HB-EGF) is an angiogenic factor mediating radial migration of the developing forebrain, while vascular endothelial growth factor (VEGF) is known to influence rostral migratory stream in rodents. Cell migratory defects have been identified in animal models of hydrocephalus; however, the relationship between HB-EGF and hydrocephalus is unclear. We show that mice overexpressing human HB-EGF with β-galactosidase reporter exhibit an elevated VEGF, localization of β-galactosidase outside the subventricular zone (SVZ), subarachnoid hemorrhage, and ventriculomegaly. In Wistar polycystic kidney rats with hydrocephalus, alteration of migratory trajectory is detected. Furthermore, VEGF infusions into the rats result in ventriculomegaly with an increase of SVZ neuroblast in rostral migratory stream, whereas VEGF ligand inhibition prevents it. Our results support the idea that excess HB-EGF leads to a significant elevation of VEGF and ventricular dilatation. These data suggest a potential pathophysiological mechanism that elevated HB-EGF can elicit VEGF induction and hydrocephalus.

Physical Weight Loading Induces Expression of Tryptophan Hydroxylase 2 in the Brain Stem

Sustaining brain serotonin is essential in mental health. Physical activities can attenuate mental problems by enhancing serotonin signaling. However, such activity is not always possible in disabled individuals or patients with dementia. Knee loading, a form of physical activity, has been found to mimic effects of voluntary exercise. Focusing on serotonergic signaling, we addressed a question: Does local mechanical loading to the skeleton elevate expression of tryptophan hydroxylase 2 (tph2) that is a rate-limiting enzyme for brain serotonin? A 5 min knee loading was applied to mice using 1 N force at 5 Hz for 1,500 cycles. A 5-min treadmill running was used as an exercise (positive) control, and a 90-min tail suspension was used as a stress (negative) control. Expression of tph2 was determined 30 min – 2 h in three brain regions ––frontal cortex (FC), ventromedial hypothalamus (VMH), and brain stem (BS). We demonstrated for the first time that knee loading and treadmill exercise upregulated the mRNA level of tph2 in the BS, while tail suspension downregulated it. The protein level of tph2 in the BS was also upregulated by knee loading and downregulated by tail suspension. Furthermore, the downregulation of tph2 mRNA by tail suspension can be partially suppressed by pre-application of knee loading. The expression of tph2 in the FC and VMH was not significantly altered with knee loading. In this study we provided evidence that peripheral mechanical loading can activate central tph2 expression, suggesting that physical cues may mediate tph2-cathalyzed serotonergic signaling in the brain.

VEGF: A potential target for hydrocephalus

Growth factors are primarily responsible for the genesis, differentiation and proliferation of cells and maintenance of tissues. Given the central role of growth factors in signaling between cells in health and in disease, it is understandable that disruption of growth factor-mediated molecular signaling can cause diverse phenotypic consequences including cancer and neurological conditions. This review will focus on the specific questions of enlarged cerebral ventricles and hydrocephalus. It is also well known that angiogenic factors, such as vascular endothelial growth factor (VEGF), affect tissue permeability through activation of receptors and adhesion molecules; hence, recent studies showing elevations of this factor in pediatric hydrocephalus led to the demonstration that VEGF can induce ventriculomegaly and altered ependyma when infused in animals. In this review, we discuss recent findings implicating the involvement of biochemical and biophysical factors that can induce a VEGF-mimicking effect in communicating hydrocephalus and pay particular attention to the role of the VEGF system as a potential pharmacological target in the treatment of some cases of hydrocephalus. The source of VEGF secretion in the cerebral ventricles, in periventricular regions and during pathologic events including hydrocephalus following hypoxia and hemorrhage is sought. The review is concluded with a summary of potential non-surgical treatments in preclinical studies suggesting several molecular targets including VEGF for hydrocephalus and related neurological disorders.

Effect of rhHB-EGF on the expression of hydrocephalus-related genes in vitro

Background Heparin binding epidermal growth factor like growth factor (HB-EGF) is a member of the epidermal growth factor family. The role of HB-EGF in cardiovascular disease such as atherosclerosis has been well-reported but its pertinence to cerebrovascular disease including hydrocephalus is unknown. In our laboratory human HB-EGF transgenic mice have been observed to demonstrate communicating hydrocephalus. The goal of this study was to find any difference in expression of three genes in vascular-lining cells under varying concentration of this growth factor.