Tanya L. Butler

Infusion of Human Umbilical Cord Blood Cells in a Rat Model of Stroke Dose-Dependently Rescues Behavioral Deficits and Reduces Infarct Volume

Background and Purpose— Intravenously delivered human umbilical cord blood cells (HUCBC) have been previously shown to improve functional recovery of stroked rats. To extend these findings, we examined the behavioral recovery and stroke infarct volume in the presence of increasing doses of HUCBC after permanent middle cerebral artery occlusion (MCAO). Methods— Rats were subjected to MCAO and allowed to recover for 24 hours before intravenous infusion of 104 up to 3 to 5×107 HUCBC. Behavioral tests (spontaneous activity, step test, elevated body swing test) were performed 1 week before MCAO and at 2 and 4 weeks after HUCBC infusion. On completion of behavioral testing, animals were euthanized and brain infarct volumes quantified. HUCBC were identified by immunofluorescence for human nuclei and by polymerase chain reaction (PCR) using primers specific for human glycerol 3-phosphate dehydrogenase. Results— At 4 weeks after infusion, there was a significant recovery in behavioral performance when 106 or more HUCBC were delivered (p=0.001 to p=0.05). Infarct volume measurements revealed an inverse relationship between HUCBC dose and damage volume, which reached significance at the higher HUCBC doses (107 cells, p<0.01; 3 to 5×107 cells, p<0.05). Moreover, HUCBC were localized by immunohistochemistry and PCR analysis only in the injured brain hemisphere and spleen. Conclusions— These results extend previous observations of HUCBC infusion in the MCAO rat stroke model by demonstrating a dose relationship between HUCBC, behavioral improvement, and neuronal sparing.

Mutations in Cardiac T-Box Factor Gene TBX20 Are Associated with Diverse Cardiac Pathologies, Including Defects of Septation and Valvulogenesis and Cardiomyopathy

The T-box family transcription factor gene TBX20 acts in a conserved regulatory network, guiding heart formation and patterning in diverse species. Mouse Tbx20 is expressed in cardiac progenitor cells, differentiating cardiomyocytes, and developing valvular tissue, and its deletion or RNA interference-mediated knockdown is catastrophic for heart development. TBX20 interacts physically, functionally, and genetically with other cardiac transcription factors, including NKX2-5, GATA4, and TBX5, mutations of which cause congenital heart disease (CHD). Here, we report nonsense (Q195X) and missense (I152M) germline mutations within the T-box DNA-binding domain of human TBX20 that were associated with a family history of CHD and a complex spectrum of developmental anomalies, including defects in septation, chamber growth, and valvulogenesis. Biophysical characterization of wild-type and mutant proteins indicated how the missense mutation disrupts the structure and function of the TBX20 T-box. Dilated cardiomyopathy was a feature of the TBX20 mutant phenotype in humans and mice, suggesting that mutations in developmental transcription factors can provide a sensitized template for adult-onset heart disease. Our findings are the first to link TBX20 mutations to human pathology. They provide insights into how mutation of different genes in an interactive regulatory circuit lead to diverse clinical phenotypes, with implications for diagnosis, genetic screening, and patient follow-up.

Neurodegeneration in the rat hippocampus and striatum after middle cerebral artery occlusion

Animal models of ischemia are in wide use to elucidate the molecular mechanisms of brain injury that result from cardiovascular disease in humans. We have used the fluorescent, anionic dye, Fluoro-Jade, to examine cellular degeneration that occurs in association with the middle cerebral artery occlusion (MCAO) model. MCAO results in cortical infarction as well as damage to the hippocampus leading to a delayed form of death of hippocampal neurons. We examined brain sections at 6 h, 12 h, 1, 4, 7, 14 and 21 days after injury. Fluoro-Jade labeling of the striatum was seen over a protracted time-course, with degeneration beginning by 6 h after injury. Neuronal degeneration in the hippocampus, in contrast, occurs between 12 h and 7 days after injury with neuronal death reaching a peak at 4 days. GFAP/Fluoro-Jade double labeling revealed that the Fluoro-Jade positive staining at late time-points in the striatum included astrocytic cells. Together, the results show Fluoro-Jade to be a useful marker of cellular degeneration following ischemic injury. Further, the use of this dye has enabled us to demonstrate previously undescribed events of cellular injury resulting from ischemia.

NF-κB protects neurons from ischemic injury after middle cerebral artery occlusion in mice

Knowledge about the molecular mechanisms of neuronal survival following ischemia is crucial to the development of therapeutic interventions for victims of stroke. Previous research in our laboratory has implicated nuclear factor-kappaB (NF-kappaB) as contributing to neuronal survival in response to toxic or ischemic brain insult, with in vivo models having focused on the rat. To take advantage of genetic alterations available in the mouse, we utilized a murine transient endovascular middle cerebral artery occlusion (MCAO) model to examine the influence of NF-kappaB on neuronal survival. When brains were immunostained for the nuclear localization sequence (NLS) of the p50 subunit of NF-kappaB, a unilateral increase in immunoreactivity was seen, especially in pyramidal cell layers of the ipsilateral (stroked) hippocampus. When transgenic mice lacking p50 were compared with non-transgenic counterparts using Fluoro-Jade, a marker for neurodegeneration, both the hippocampus and striatum showed enhanced neurodegeneration at various survival times after 1 h of MCAO. In the hippocampus specifically, there was an eightfold increase in Fluoro-jade staining in the p50 knockout group vs. the non-transgenic group. Sections double stained for Fluoro-Jade and NF-kappaB activity (using a mouse engineered with a NF-kappaB responsive promoter driving a LacZ gene to produce beta galactosidase) demonstrated neuronal degeneration only in regions sparsely showing NF-kappaB activity, and those demonstrating NF-kappaB activity failed to degenerate. These data provide evidence that NF-kappaB participates in survival signaling following temporary focal ischemia, and thus may represent an attractive target for pharmacologic activation in the treatment of stroke.

A Novel Class of Anticancer Compounds Targets the Actin Cytoskeleton in Tumor Cells

The actin cytoskeleton is a potentially vulnerable property of cancer cells, yet chemotherapeutic targeting attempts have been hampered by unacceptable toxicity. In this study, we have shown that it is possible to disrupt specific actin filament populations by targeting isoforms of tropomyosin, a core component of actin filaments, that are selectively upregulated in cancers. A novel class of anti-tropomyosin compounds has been developed that preferentially disrupts the actin cytoskeleton of tumor cells, impairing both tumor cell motility and viability. Our lead compound, TR100, is effective in vitro and in vivo in reducing tumor cell growth in neuroblastoma and melanoma models. Importantly, TR100 shows no adverse impact on cardiac structure and function, which is the major side effect of current anti-actin drugs. This proof-of-principle study shows that it is possible to target specific actin filament populations fundamental to tumor cell viability based on their tropomyosin isoform composition. This improvement in specificity provides a pathway to the development of a novel class of anti-actin compounds for the potential treatment of a wide variety of cancers.

Temporary focal ischemia in the mouse: Technical aspects and patterns of Fluoro-Jade evident neurodegeneration

Animal models of cerebral infarction are crucial to understanding the mechanisms of neuronal survival following ischemic brain injury and to the development of therapeutic interventions for victims of all types of stroke. Rodents have been used extensively in such research. One rodent model of stroke utilizes either permanent or temporary occlusion of the middle cerebral artery (MCAO) to produce ischemia. Since the development of an endovascular method for this was published in 1989, MCAO has been applied commonly to the rat, and often paired with 2, 3, 5-triphenyltetrazolium chloride (TTC) staining for stroke volume measurement. Meanwhile, advances in the ability to genetically alter mice have allowed exciting lines of research into ischemia. Because of technical demands and issues with survival, relatively few laboratories have investigated the MCAO method in the mouse. Our present work utilizes a mouse middle cerebral occlusion (MCAO) model of embolic stroke to study neuronal degeneration following temporary focal cerebral ischemia. C57Bl/6J mice were used to examine the exact effects of MCAO using Fluoro-Jade, a marker of neurodegeneration that allows observation of specific brain regions and cells destined to die. A time course of escalating neuronal degeneration from 10 min to 7 days following MCAO was established. Technical aspects of this popular method for transient focal ischemia as it applies to the mouse are discussed.

Increased connective tissue growth factor associated with cardiac fibrosis in the mdx mouse model of dystrophic cardiomyopathy

Cardiomyopathy contributes to morbidity and mortality in Duchenne muscular dystrophy (DMD), a progressive muscle‐wasting disorder. A major feature of the hearts of DMD patients and the mdx mouse model of the disease is cardiac fibrosis. Connective tissue growth factor (CTGF) is involved in the fibrotic process in many organs. This study utilized the mdx mouse model to assess the role of CTGF and other extracellular matrix components during the development of fibrosis in the dystrophic heart. Left ventricular function of mdx and control mice at 6, 29 and 43 weeks was measured by echocardiography. Young (6 weeks old) mdx hearts had normal function and histology. At 29 weeks of age, mdx mice developed cardiac fibrosis and increased collagen expression. The onset of fibrosis was associated with increased CTGF transcript and protein expression. Increased intensity of CTGF immunostaining was localized to fibrotic areas in mdx hearts. The upregulation of CTGF was also concurrent with increased expression of tissue inhibitor of matrix metalloproteinases (TIMP‐1). These changes persisted in 43 week old mdx hearts and were combined with impaired cardiac function and increased gene expression of transforming growth factor (TGF)‐β1 and matrix metalloproteinases (MMP‐2, MMP‐9). In summary, an association was observed between cardiac fibrosis and increased CTGF expression in the mdx mouse heart. CTGF may be a key mediator of early and persistent fibrosis in dystrophic cardiomyopathy.

GATA4 mutations in 357 unrelated patients with congenital heart malformation.

Congenital heart disease (CHD) represents one of the most common birth defects, but the genetic causes remain largely unknown. Mutations in GATA4, encoding a zinc finger transcription factor with a pivotal role in heart development, have been associated with CHD in several familial cases and a small subset of sporadic patients. To estimate the pathogenetic role of GATA4 in CHD, we screened for mutations in 357 unrelated patients with different congenital heart malformations. In addition to nine synonymous changes, we identified two known (A411V and D425N) and two novel putative mutations (G69D and P163R) in five patients with atrial or ventricular septal defects that were not seen in control subjects. The four mutations did not show altered GATA4 transcriptional activity in synergy with the transcription factors NKX2-5 and TBX20. Our data expand the spectrum of mutations associated with cardiac septal defects but do not support GATA4 mutations as a common cause of CHD.

Somatic mutations in NKX2–5, GATA4, and HAND1 are not a common cause of tetralogy of Fallot or hypoplastic left heart

The majority of congenital heart disease (CHD) occurs as a sporadic finding, with a minority of cases associated with a known genetic abnormality. Combinations of genetic and environmental factors are implicated, with the recent and intriguing hypothesis that an apparently high rate of somatic mutations might explain some sporadic CHD. We used samples of right ventricular myocardium from patients undergoing surgical repair of tetralogy of Fallot (TOF) and hypoplastic left heart (HLH) to examine the incidence of somatic mutation in cardiac tissue. TOF is a common form of cyanotic CHD, occurring in 3.3 per 10,000 live births. HLH is a rare defect in which the left side of the heart is severely under‐developed. Both are severe malformations whose genetic etiology is largely unknown. We carried out direct sequence analysis of the NKX2–5 and GATA4 genes from fresh frozen cardiac tissues and matched blood samples of nine TOF patients. Analysis of NKX2–5, GATA4, and HAND1 was performed from cardiac tissue of 24 HLH patients and three matched blood samples. No somatic or germline mutations were identified in the TOF or HLH patients. Although limited by sample size, our study suggests that somatic mutations in NKX2–5 and GATA4 are not a common cause of isolated TOF or HLH.

Myocardial ischemia is more important than the effects of cardiopulmonary bypass on myocardial water handling and postoperative dysfunction: A pediatric animal model

OBJECTIVES Low cardiac output state is the principal cause of morbidity after surgical intervention for congenital heart disease. Myocardial ischemia-reperfusion injury, apoptosis, capillary leak syndrome, and myocardial edema are associated factors. We established a clinically relevant model to examine relationships between myocardial ischemia, edema, and cardiac dysfunction and to assess the role of the water transport proteins aquaporins. METHODS Sixteen lambs were studied. Seven were control animals not undergoing cardiopulmonary bypass, and 9 underwent bypass. Six had 90 minutes of aortic crossclamping with blood cardioplegia and moderate hypothermia. The remaining 3 underwent cardiopulmonary bypass without aortic crossclamping. Hemodynamic and biochemical data were recorded, and myocardial edema, apoptotic markers, and aquaporin expression were determined after death. RESULTS The group undergoing cardiopulmonary bypass with aortic crossclamping had a low cardiac output state, with early postoperative tachycardia, hypotension, increased serum lactate levels, and impaired tissue oxygen delivery (P < .05) compared with the group undergoing cardiopulmonary bypass without aortic crossclamping. The lambs undergoing cardiopulmonary bypass with aortic crossclamping had increased myocardial water (P < .05) compared with those not undergoing cardiopulmonary bypass and a 2-fold increase in aquaporin 1 mRNA expression (P < .05) compared with those not undergoing cardiopulmonary bypass and those undergoing cardiopulmonary bypass without aortic crossclamping. CONCLUSIONS A temporal association between hemodynamic dysfunction, myocardial edema, and increased aquaporin 1 expression was demonstrated. Cardiopulmonary bypass without ischemia was associated with minimal edema, negligible myocardial dysfunction, and static aquaporin expression. Ischemic reperfusion injury is the main cause of myocardial edema and myocardial dysfunction, but a causal relationship between edema and dysfunction remains to be proved.