Faculty Bibliography

The transcription factor hypoxia-inducible factor 1-alpha (HIF-1alpha) is responsible for the downstream expression of over 60 genes that regulate cell survival and metabolism in hypoxic conditions as well as those that enhance angiogenesis to alleviate hypoxia. However, under normoxic conditions, HIF-1alpha is hydroxylated by prolyl hydroxylase 2, and subsequently degraded, with a biological half-life of less than five minutes. Here we investigated the therapeutic potential of inhibiting HIF-1alpha degradation through short hairpin RNA silencing of PHD-2 in the setting of diabetic wounds and limb ischemia. Treatment of diabetic mouse fibroblasts with shPHD-2 in vitro resulted in decreased levels of PHD-2 transcript demonstrated by qRT-PCR, higher levels of HIF-1alpha as measured by western blot, and higher expression of the downstream angiogenic genes SDF-1 and VEGFalpha, as measured by qRT-PCR. In vivo, shPHD-2 accelerated healing of full thickness excisional wounds in diabetic mice compared to shScr control, (14.33 +/- 0.45 days vs. 19 +/- 0.33 days) and was associated with an increased vascular density. Delivery of shPHD-2 also resulted in improved perfusion of ischemic hind limbs compared to shScr, prevention of distal digit tip necrosis, and increased survival of muscle tissue. Knockdown of PHD-2 through shRNA treatment has the potential to stimulate angiogenesis through overexpression of HIF-1alpha and upregulation of pro-angiogenic genes downstream of HIF-1alpha, and may represent a viable, non-viral approach to gene therapy for ischemia related applications.

Recent developments in tissue clearing methods have provided investigators with an invaluable tool for visualizing and mapping three dimensional macromolecular structures and processes. By implementing a routine protocol, based on the passive clarity technique (PACT) method, for tissue clearing, the Research Histopathology Core at NYU Langone Medical Center seeks to provide investigators with a reliable, customizable service in conjunction with the immunohistochemistry (IHC) and Microscopy Cores in order to produce results in the most efficient way possible for both the investigators and the cores. The PACT method of clearing allows visualization of endogenous fluorescence and immunofluorescence labeling performed by the Core, or both. Stabilization through transparent hydrogel cross-linking, followed by delipidation in an sodium dodecyl sulfate buffer, results in a clear tissue sample that remains structurally sound with proteins, nucleic acids, and any associated labels in place. The clearing buffer can also be modified to allow simultaneous decalcification of bone specimens. Final clearing is achieved in a refractive index matching solution (RIMS buffer) which also serves as the microscopy medium. Cleared and labeled tissue can then be imaged on the Microscopy Core's Zeiss lightsheet microscope, allowing multichannel fluorescence from a range of angles and Z-stacking. The lightsheet microscope excites and detects only one thin optical section of the specimen at a time, making three dimensional imaging exceptionally light efficient. By honing proficiency in tissue clearing via the PACT method and working in close collaboration with neighboring core labs, the Histopathology Core can increase its breadth of expertise while relieving investigators of the time and cost intensive burden of protocol development and training.

Atopic dermatitis (AD) is a chronic, inflammatory skin condition caused by a complex interaction of genetic and environment factors. Inherited defects within the stratum corneum are increasingly recognised as a key causative factor in the development of AD. Patients with AD also demonstrate a high rate of colonisation by microbes, notably Staphylococcus aureus. Controversy exists regarding the use of antimicrobial agents in the management of AD. We briefly review the role of stratum corneum dysfunction in AD, the influence of cutaneous colonisation and infections, and provide an update on the utility of anti-staphylococcal treatment in AD.

BACKGROUND/AIMS/OBJECTIVE:The fact that ouabain has been identified as an endogenous substance, led us to inquire its physiological role in epithelial cells. Based on previous observations, we hypothesized that it influences processes related to cell contacts. Previously we have shown that nanomolar concentrations of ouabain up-regulate tight junctions, accelerate ciliogenesis, and increase gap junctional intercellular communication (GJIC). Given that silencing assays indicated that connexin 43 (Cnx43) is involved in the GJIC response, in the present work we study whether ouabain affects Cnx43 expression and distribution. METHODS:We seeded confluent monolayers of epithelial renal MDCK cells and incubated them with 10 nM ouabain during 1 h. Then we measured, by densitometric analysis of Western blot assays, the amount of Cnx43 in cells and in fractions enriched of plasma membrane. We also studied its localization with immunofluorescence and confocal microscopy. RESULTS:Cnx43 is remarkably displayed, outlining the borders of cells gathered in clusters, randomly scattered throughout the monolayer. Ouabain increases the density of such clusters, as well as the average number of cells per cluster, without inducing the synthesis of new Cnx43. It also promotes relocation towards the membrane, of subunits already available. The fact that such changes are inhibited by PP2 and PD98059 indicates that a signaling pathway, that includes c-Src and ERK1/2, is involved in this response. CONCLUSION/CONCLUSIONS:Ouabain induces the translocation of Cnx43 from the cytoplasm to the plasma membrane. These findings support our hypothesis that one of the physiological roles of ouabain is the modulation of physiological processes that depend on cell to cell contacts.

We investigated the contribution of blood vessel formation and neuronal excitability to the development of functional neural circuitry in larval zebrafish by analyzing oculomotor performance in response to visual and vestibular stimuli. To address the dependence of neuronal function on the presence of blood vessels, we compared wild type embryos to reck and cloche mutants that lacked intracerebral blood vessels. To test how neuronal excitability impacts neuronal development and intracerebral vascularization, we blocked neural activity using Tetraodotoxin (TTX) and Tricaine. In reck mutants, we found both slow phase horizontal tracking and fast phase resets with only a slightly reduced amplitude and bandwidth. Spontaneous saccades, eye position holding and vestibular gravitoinertial induced eye rotation were also present. All of these behaviors except for visual tracking were observed in cloche mutants that lacked any head vasculature. Thus, numerous oculomotor neuronal circuits spanning the forebrain, midbrain and hindbrain compartments, ending in motor innervations of the eye muscles, were correctly formed and generated appropriate oculomotor behaviors without blood vessels. However, our observations indicate that beginning at approximately six days, circulation was required for sustained behavioral performance. We further found that blocking neuronal excitability with either TTX or Tricaine up to 4-5 days post fertilization did not noticeably interfere with intracerebral blood vessel formation in wild type larvae. After removal of drug treatments, the oculomotor behaviors returned within hours. Thus, development of neuronal circuits that drive oculomotor performance does not require neuronal spiking or activity. Together these findings demonstrate that neither vascularization nor neuronal excitability are essential for the formation of numerous oculomotor nuclei with intricately designed connectivity and signal processing. We conclude that a genetic blueprint specifies early larval structural and physiological features, and this developmental strategy may be viewed as a unique adaptation required for early survival.