Julie A. Wolfram

Keratinocyte but Not Endothelial Cell-Specific Overexpression of Tie2 Leads to the Development of Psoriasis

Psoriasis is initiated and maintained through a multifaceted interplay between keratinocytes, blood vessels, gene expression, and the immune system. One previous psoriasis model demonstrated that overexpression of the angiopoietin receptor Tie2 in endothelial cells and keratinocytes led to the development of a psoriasiform phenotype; however, the etiological significance of overexpression in each cell type alone was unclear. We have now engineered two new mouse models whereby Tie2 expression is confined to either endothelial cells or keratinocytes. Both lines of mice have significant increases in dermal vasculature but only the KC-Tie2-overexpressing mice developed a cutaneous psoriasiform phenotype. These mice spontaneously developed characteristic hallmarks of human psoriasis, including extensive acanthosis, increases in dermal CD4(+) T cells, infiltrating epidermal CD8(+) T cells, dermal dendritic cells and macrophages, and increased expression of cytokines and chemokines associated with psoriasis, including interferon-gamma, tumor necrosis factor-alpha, and interleukins 1alpha, 6, 12, 22, 23, and 17. Host-defense molecules, cathelicidin, beta-defensin, and S100A8/A9, were also up-regulated in the hyperproliferative skin. All of the phenotypic traits were completely reversed without any scarring following repression of the transgene and were significantly improved following treatment with the anti-psoriasis systemic therapeutic, cyclosporin A. Therefore, confining Tie2 overexpression solely to keratinocytes results in a mouse model that meets the clinical, histological, immunophenotypic, biochemical, and pharmacological criteria required for an animal model of human psoriasis.

Depletion of antigen-presenting cells by clodronate liposomes reverses the psoriatic skin phenotype in KC-Tie2 mice.

Background  There is ongoing debate regarding the initiation of psoriatic plaque as primarily arising from an anomaly in epidermal keratinocytes (KCs) or from abnormalities in immunocytes that secondarily activate otherwise normal KCs. In mice engineered to overexpress the angiopoietin receptor Tie2 in KCs, skin spontaneously develops the characteristic clinical, histological and immune cell phenotypes of psoriasis which can be reversed with either transgene repression or ciclosporin administration, suggesting key roles for both KCs and T cells in mediating the skin disease in this murine model.

Gene Therapy to Treat Cardiovascular Disease

Coronary artery disease, heart failure, and cardiac arrhythmias are major causes of morbidity and mortality in the United States. The overall death rate for all cardiovascular diseases was 236.1 per 100 000 persons, accounting for 1 of every 3 deaths in America.[1][1] Cardiac diseases can affect

Cell Cycle Re-Entry and Mitochondrial Defects in Myc- Mediated Hypertrophic Cardiomyopathy and Heart Failure

While considerable evidence supports the causal relationship between increases in c-Myc (Myc) and cardiomyopathy as a part of a “fetal re-expression” pattern, the functional role of Myc in mechanisms of cardiomyopathy remains unclear. To address this, we developed a bitransgenic mouse that inducibly expresses Myc under the control of the cardiomyocyte-specific MHC promoter. In adult mice the induction of Myc expression in cardiomyocytes in the heart led to the development of severe hypertrophic cardiomyopathy followed by ventricular dysfunction and ultimately death from congestive heart failure. Mechanistically, following Myc activation, cell cycle markers and other indices of DNA replication were significantly increased suggesting that cell cycle-related events might be a primary mechanism of cardiac dysfunction. Furthermore, pathological alterations at the cellular level included alterations in mitochondrial function with dysregulation of mitochondrial biogenesis and defects in electron transport chain complexes I and III. These data are consistent with the known role of Myc in several different pathways including cell cycle activation, mitochondrial proliferation, and apoptosis, and indicate that Myc activation in cardiomyocytes is an important regulator of downstream pathological sequelae. Moreover, our findings indicate that the induction of Myc in cardiomyocytes is sufficient to cause cardiomyopathy and heart failure, and that sustained induction of Myc, leading to cell cycle re-entry in adult cardiomyocytes, represents a maladaptive response for the mature heart.

Altered hypoxia-inducible factor-1 alpha expression levels correlate with coronary vessel anomalies.

The outflow tract myocardium and other regions corresponding to the location of the major coronary vessels of the developing chicken heart, display a high level of hypoxia as assessed by the hypoxia indicator EF5. The EF5‐positive tissues were also specifically positive for nuclear‐localized hypoxia inducible factor‐1 alpha (HIF‐1α), the oxygen‐sensitive component of the hypoxia inducible factor‐1 (HIF‐1) heterodimer. This led to our hypothesis that there is a “template” of hypoxic tissue that determines the stereotyped pattern of the major coronary vessels. In this study, we disturbed this template by altering ambient oxygen levels (hypoxia 15%; hyperoxia 75–40%) during the early phases of avian coronary vessel development, in order to alter tissue hypoxia, HIF‐1α protein expression, and its downstream target genes without high mortality. We also altered HIF‐1α gene expression in the embryonic outflow tract cardiomyocytes by injecting an adenovirus containing a constitutively active form of HIF‐1α (AdCA5). We assayed for coronary anomalies using anti‐alpha‐smooth muscle actin immunohistology. When incubated under abnormal oxygen levels or injected with a low titer of the AdCA5, coronary arteries displayed deviations from their normal proximal connections to the aorta. These deviations were similar to known clinical anomalies of coronary arteries. These findings indicated that developing coronary vessels may be subject to a level of regulation that is dependent on differential oxygen levels within cardiac tissues and subsequent HIF‐1 regulation of gene expression. Developmental Dynamics 238:2688–2700, 2009.

Cutaneous manipulation of vascular growth factors leads to alterations in immunocytes, blood vessels and nerves: Evidence for a cutaneous neurovascular unit.

BACKGROUND Skin cells produce soluble factors which influence keratinocyte proliferation, angiogenesis, nerve innervation and immunocyte response. OBJECTIVE To test the hypothesis that epidermal-dermal interactions influence neural outgrowth, vascular survival, immunocyte recruitment and keratinocyte proliferation. METHODS We genetically manipulated the epidermis to express excess vascular endothelial growth factor (VEGF) and/or angiopoietin-1 (Ang1) and then examined the epidermal and dermal phenotypes. We compared these findings with those occurring following overexpression of the Ang1 receptor Tie2 in endothelial cells or keratinocytes. RESULTS Keratinocyte-overexpression of Ang1 resulted in increased epidermal thickness compared to control littermates. Keratinocyte-specific overexpression of Ang1 or VEGF increased dermal angiogenesis compared to control animals and combined Ang1-VEGF lead to further increases. Cutaneous leukocyte examination revealed increases in CD4(+) T cell infiltration in mice with keratinocyte-specific overexpression of Ang1, VEGF and Ang1-VEGF combined; in contrast only keratinocyte-specific Ang1 overexpression increased cutaneous F4/80(+) macrophage numbers. Interestingly, combined keratinocyte-derived Ang1-VEGF overexpression reduced significantly the number of F4/80(+) and Cd11c(+) cells compared to mice overexpressing epidermal Ang1 alone. Endothelial cell-specific Tie2 overexpression increased dermal angiogenesis but failed to influence the epidermal and immune cell phenotypes. Keratinocyte-specific Tie2 expressing mice had the highest levels of CD4(+), CD8(+) and CD11c(+) cell numbers and acanthosis compared to all animals. Finally, increases in the number of cutaneous nerves were found in all transgenic mice compared to littermate controls. CONCLUSION These findings demonstrate that change to one system (vascular or epidermal) results in change to other cutaneous systems and suggest that individual molecules can exert effects on multiple systems.

Accumulation of intraneuronal amyloid-β is common in normal brain.

Intraneuronal amyloid-β (iAβ) accumulation has been demonstrated in Alzheimer disease (AD). Although extracellular amyloid plaques composed primarily of aggregated amyloid-β are one of the main pathological features of AD, functional characterization of iAβ is still lacking. In this study, we identified the normal distribution of iAβ through an analysis of hippocampal sections from a series of over 90 subjects with diverse antemortem clinical findings. In addition to AD cases, iAβ in pyramidal neurons was readily and reproducibly demonstrated in the majority of control cases. Similar findings for controls were made across all ages, spanning from infants to the elderly. There was no correlation of iAβ between gender, postmortem interval, or age. While the possible pathophysiological significance of iAβ accumulation in AD remains to be elucidated, careful examination of iAβ found in the normal brain may be informative for determining the biological role of iAβ and how this function changes during disease. Current findings support a physiological role for iAβ in neuronal function over the entire lifespan.

Therapeutic potential of c-Myc inhibition in the treatment of hypertrophic cardiomyopathy

Investigating the pathophysiological importance of the molecular and mechanical development of cardiomyopathy is critical to find new and broader means of protection against this disease that is increasing in prevalence and impact. The current available treatment options for cardiomyopathy mainly focus on treating symptoms and strive to make the patient more comfortable while preventing progression of disease and sudden death. The proto-oncogene c-Myc (Myc) has been shown to be increased in many different types of heart disease, including hypertrophic cardiomyopathy, before any signs of the disease are present. As the mechanisms of action and multiple pathways of dependent actions of Myc are being dissected by many research groups, inhibition of Myc is becoming an attractive paradigm for prevention and treatment of cardiomyopathy and heart failure. Elucidating the role Myc plays in the development, propagation and perpetuation of cardiomyopathy and heart failure will one day translate into potential therapeutics for cardiomyopathy.

BRG1 and BRM function antagonistically with c-MYC in adult cardiomyocytes to regulate conduction and contractility

RATIONALE The contractile dysfunction that underlies heart failure involves perturbations in multiple biological processes ranging from metabolism to electrophysiology. Yet the epigenetic mechanisms that are altered in this disease state have not been elucidated. SWI/SNF chromatin-remodeling complexes are plausible candidates based on mouse knockout studies demonstrating a combined requirement for the BRG1 and BRM catalytic subunits in adult cardiomyocytes. Brg1/Brm double mutants exhibit metabolic and mitochondrial defects and are not viable although their cause of death has not been ascertained. OBJECTIVE To determine the cause of death of Brg1/Brm double-mutant mice, to test the hypothesis that BRG1 and BRM are required for cardiac contractility, and to identify relevant downstream target genes. METHODS AND RESULTS A tamoxifen-inducible gene-targeting strategy utilizing αMHC-Cre-ERT was implemented to delete both SWI/SNF catalytic subunits in adult cardiomyocytes. Brg1/Brm double-mutant mice were monitored by echocardiography and electrocardiography, and they underwent rapidly progressive ventricular dysfunction including conduction defects and arrhythmias that culminated in heart failure and death within 3weeks. Mechanistically, BRG1/BRM repressed c-Myc expression, and enforced expression of a DOX-inducible c-MYC trangene in mouse cardiomyocytes phenocopied the ventricular conduction defects observed in Brg1/Brm double mutants. BRG1/BRM and c-MYC had opposite effects on the expression of cardiac conduction genes, and the directionality was consistent with their respective loss- and gain-of-function phenotypes. To support the clinical relevance of this mechanism, BRG1/BRM occupancy was diminished at the same target genes in human heart failure cases compared to controls, and this correlated with increased c-MYC expression and decreased CX43 and SCN5A expression. CONCLUSION BRG1/BRM and c-MYC have an antagonistic relationship regulating the expression of cardiac conduction genes that maintain contractility, which is reminiscent of their antagonistic roles as a tumor suppressor and oncogene in cancer.

Impact of Insurance Precertification on Discharge of Stroke Patients to Acute Rehabilitation or Skilled Nursing Facility

BACKGROUND AND PURPOSE The purpose of this study is to determine if the common insurance practice of requiring precertification before a medically ready stroke patient can be discharged to a skilled nursing facility (SNF) or inpatient rehabilitation facility (IRF) causes a delay in discharge. Eliminating delays in discharge of stroke patients is important given the increasing demands for health-care efficiency after the passage of the Affordable Health Care Act. METHODS A retrospective chart review of 1007 patients who were admitted to our comprehensive stroke center with the primary diagnosis of stroke over a 12-month period was performed. Out of the patient pool, 289 patients met the inclusion criterion of a primary diagnosis of stroke that required discharge to a SNF or IRF. All 289 patients were medically cleared for discharge to a SNF or IRF by a board-certified vascular neurologist. RESULTS Of the 289 patients who met the inclusion criteria, 118 required insurance precertification and 171 did not require precertification before being discharged to a SNF or IRF. All 118 patients who required precertification had private health insurance. The patients who required insurance precertification had an average delay of discharge (DOD) of 1.5 days, and those patients who did not require precertification had an average DOD of .8 days (P value <.0001). After removing the outliers, the difference in the length of stay (LOS) between the 2 groups became statistically significant (P value < .04). CONCLUSION The results of this study demonstrate that insurance precertification leads to delay in discharge, increased LOS, and increased hospital costs for stroke patients.