Dirk M. Hentschel

TRIP-Br: a novel family of PHD zinc finger- and bromodomain-interacting proteins that regulate the transcriptional activity of E2F-1/DP-1

We report the isolation of TRIP‐Br1, a transcriptional regulator that interacts with the PHD‐bromodomain of co‐repressors of Krüppel‐associated box (KRAB)‐mediated repression, KRIP‐1(TIF1β) and TIF1α, as well as the co‐activator/adaptor p300/CBP. TRIP‐Br1 and the related protein TRIP‐Br2 possess transactivation domains. Like MDM2, which has a homologous transactivation domain, TRIP‐Br proteins functionally contact DP‐1, stimulating E2F‐1/DP‐1 transcriptional activity. KRIP‐1 potentiates TRIP‐Br protein co‐activation of E2F‐1/DP‐1. TRIP‐Br1 is a component of a multiprotein complex containing E2F‐1 and DP‐1. Co‐expression of the retinoblastoma gene product (RB) abolishes baseline E2F‐1/DP‐1 transcriptional activity as well as TRIP‐Br/KRIP‐1 co‐activation, both of which are restored by the adenovirus E1A oncoprotein. These features suggest that TRIP‐Br proteins function at E2F‐responsive promoters to integrate signals provided by PHD‐ and/or bromodomain‐ containing transcription factors. TRIP‐Br1 is identical to the cyclin‐dependent kinase 4 (cdk4)‐binding protein p34SEI‐1, which renders the activity of cyclin D/cdk4 resistant to the inhibitory effect of p16INK4a during late G1. TRIP‐Br1(p34SEI‐1) is differentially overexpressed during the G1 and S phases of the cell cycle, consistent with a dual role for TRIP‐Br1(p34SEI‐1) in the regulation of cell cycle progression through sequential effects on the transcriptional activity of E2F‐responsive promoters during G1 and S phases.

Shape effect of carbon nanovectors on angiogenesis.

Physically diverse carbon nanostructures are increasingly being studied for potential applications in cancer chemotherapy. However, limited knowledge exists on the effect of their shape in tuning the biological outcomes when used as nanovectors for drug delivery. In this study, we evaluated the effect of doxorubicin-conjugated single walled carbon nanotubes (CNT-Dox) and doxorubicin-conjugated spherical polyhydroxylated fullerenes or fullerenols (Ful-Dox) on angiogenesis. We report that CNTs exert a pro-angiogenic effect in vitro and in vivo. In contrast, the fullerenols or doxorubicin-conjugated fullerenols exerted a dramatically opposite antiangiogenic activity in zebrafish and murine tumor angiogenesis models. Dissecting the angiogenic phenotype into discrete cellular steps revealed that fullerenols inhibited endothelial cell proliferation, while CNTs attenuated the cytotoxic effect of doxorubicin on the endothelial cells. Interestingly, CNT promoted endothelial tubulogenesis, a late step during angiogenesis. Further, mechanistic studies revealed that CNTs, but not fullerenols, induced integrin clustering and activated focal adhesion kinase and downstream phosphoinositide-3-kinase (PI3K) signaling in endothelial cells, which can explain the distinct angiogenic outcomes. The results of the study highlight the function of physical parameters of nanoparticles in determining their activity in biological settings.

Nanoparticle-mediated targeting of phosphatidylinositol-3-kinase signaling inhibits angiogenesis

ObjectiveDysregulation of the phosphatidylinositol-3-kinase (PI3K) signaling pathway is a hallmark of human cancer, occurring in a majority of tumors. Activation of this pathway is critical for transformation and also for the angiogenic switch, which is a key step for tumor progression. The objective of this study was to engineer a PI3K inhibitor-loaded biodegradable nanoparticle and to evaluate its efficacy.Methods and resultsHere we report that a nanoparticle-enabled targeting of the PI3K pathway results in inhibition of downstream Akt phosphorylation, leading to inhibition of proliferation and induction of apoptosis of B16/F10 melanoma. It, however, failed to exert a similar activity on MDA-MB-231 breast cancer cells, resulting from reduced internalization and processing of nanoparticles in this cell line. Excitingly, the nanoparticle-enabled targeting of the PI3K pathway resulted in inhibition of endothelial cell proliferation and tubulogenesis, two key steps in tumor angiogenesis. Furthermore, it inhibited both B16/F10- and MDA-MB-231-induced angiogenesis in a zebrafish tumor xenotransplant model.ConclusionOur study, for the first time, shows that targeting of the PI3K pathway using nanoparticles can offer an attractive strategy for inhibiting tumor angiogenesis.

odd skipped related1 reveals a novel role for endoderm in regulating kidney versus vascular cell fate.

The kidney and vasculature are intimately linked both functionally and during development, when nephric and blood/vascular progenitor cells occupy adjacent bands of mesoderm in zebrafish and frog embryos. Developmental mechanisms that underlie the differentiation of kidney versus blood/vascular lineages remain unknown. The odd skipped related1 (osr1) gene encodes a zinc-finger transcription factor that is expressed in the germ ring mesendoderm and subsequently in the endoderm and intermediate mesoderm, prior to the expression of definitive kidney or blood/vascular markers. Knockdown of osr1 in zebrafish embryos resulted in a complete, segment-specific loss of anterior kidney progenitors and a compensatory increase in the number of angioblast cells in the same trunk region. Histology revealed a subsequent absence of kidney tubules, an enlarged cardinal vein and expansion of the posterior venous plexus. Altered kidney versus vascular development correlated with expanded endoderm development in osr1 knockdowns. Combined osr1 loss of function and blockade of endoderm development by knockdown of sox32/casanova rescued anterior kidney development. The results indicate that osr1 activity is required to limit endoderm differentiation from mesendoderm; in the absence of osr1, excess endoderm alters mesoderm differentiation, shifting the balance from kidney towards vascular development.

Coupling growth-factor engineering with nanotechnology for therapeutic angiogenesis

Therapeutic angiogenesis is an emerging paradigm for the management of ischemic pathologies. Proangiogenic Therapy is limited, however, by the current inability to deliver angiogenic factors in a sustained manner at the site of pathology. In this study, we investigated a unique nonglycosylated active fragment of hepatocyte growth factor/scatter factor, 1K1, which acts as a potent angiogenic agent in vitro and in a zebrafish embryo and a murine matrigel implant model. Furthermore, we demonstrate that nanoformulating 1K1 for sustained release temporally alters downstream signaling through the mitogen activated protein kinase pathway, and amplifies the angiogenic outcome. Merging protein engineering and nanotechnology offers exciting possibilities for the treatment of ischemic disease, and furthermore allows the selective targeting of downstream signaling pathways, which translates into discrete phenotypes.

Endothelial function predicts positive arterial-venous fistula remodeling in subjects with stage IV and V chronic kidney disease

Purpose The maturation of an arteriovenous fistula (AVF) requires remodeling of the arterial inflow and the venous outflow limbs to sustain flows sufficient to support hemodialysis. However, factors influencing remodeling of AVF are poorly understood. We hypothesized that AVF remodeling was an endothelium-dependent process. Methods This is a prospective cohort study of patients (n=25) undergoing autologous AVF formation. Brachial artery vasoreactivity studies were performed pre-operatively to assess endothelium-dependent, flow-mediated vasodilation (FMD). High-resolution ultrasound was used to assess venous and arterial diameters intraoperatively, and at 3 months. Results The mean age was 64.5 ± 13.6 yrs. Twelve patients (48%) had diabetes. The mean FMD for the entire cohort was (mean ± SEM) 5.82 ± 0.9%, (range) 0–17.3%. The vein increased in size 3.19 ± .28 to 6.11 ± .41 mm, 108.4 ± 17.9%, p=.0001, while the artery increased from 3.29 ± .14 to 4.48 ± .30 mm, 20.47 ± 10.8%, p=.013. There was a significant positive correlation between the degree of arterial and venous remodeling, r=.52, p=.023. Brachial artery FMD most strongly correlated with the magnitude of arterial remodeling, r=.47, p=.038. Patients with diabetes failed to undergo venous remodeling to the same extent as did those without diabetes, 59.2 ± 24.4% vs. 141.5 ± 25.4%, p=.04. Conclusion Impairment of endothelial function is associated with decreased arterial remodeling and final venous lumen diameter attained at 3 months. Further investigation is needed to determine whether modulation of endothelial function in this cohort can improve AVF maturation.

APC16 is a conserved subunit of the anaphase-promoting complex/cyclosome

Error-free chromosome segregation depends on timely activation of the multi-subunit E3 ubiquitin ligase APC/C. Activation of the APC/C initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for destruction. The APC/C is the principle target of the mitotic checkpoint, which prevents segregation while chromosomes are unattached to spindle microtubules. We now report the identification and characterization of APC16, a conserved subunit of the APC/C. APC16 was found in association with tandem-affinity-purified mitotic checkpoint complex protein complexes. APC16 is a bona fide subunit of human APC/C: it is present in APC/C complexes throughout the cell cycle, the phenotype of APC16-depleted cells copies depletion of other APC/C subunits, and APC16 is important for APC/C activity towards mitotic substrates. APC16 sequence homologues can be identified in metazoans, but not fungi, by four conserved primary sequence stretches. We provide evidence that the C. elegans gene K10D2.4 and the D. rerio gene zgc:110659 are functional equivalents of human APC16. Our findings show that APC/C is composed of previously undescribed subunits, and raise the question of why metazoan APC/C is molecularly different from unicellular APC/C.

Cardiovascular Implantable Electronic Device Leads in CKD and ESRD Patients: Review and Recommendations for Practice

Cardiovascular implantable electronic devices (CIEDs) are frequently utilized for management of cardiac dysrhythmias in patients with chronic kidney disease or end‐stage renal disease receiving hemodialysis. The survival benefit from use of implantable cardioverter defibrillators in patients with CKD or ESRD is not as clear as in the general population, particularly when used for primary prevention of sudden cardiac death. Transvenous CIED leads are associated with central vein stenosis resulting in significant adverse consequences for existing or future arteriovenous access. Venous hypertension from CIED lead‐related central vein stenosis is a challenging clinical problem and may require repeated percutaneous interventions, replacement of the CIED, or creation of alternative arteriovenous access. Infections associated with transvenous CIED leads are more frequent and associated with worse outcomes in patients with renal disease. Epicardial CIED leads or other nontransvenous devices may reduce complications of both central venous stenosis and endovascular infection in these vulnerable patients. Consensus recommendations are offered for avoidance and management of complications arising from the use of CIEDs and arteriovenous hemodialysis access.

Novel Non-rodent Models of Kidney Disease

Kidney disease in the 21(st) century affects increasing numbers of individuals. We continue to be challenged by our lack of understanding of the pathophysiology of acute and chronic renal disease including genetic diseases involving the kidney. Rodent knockout animals or inbred strains have greatly contributed to our understanding of many monogenetic and complex diseases. Non-rodent animal models of disease have become more attractive since genomic data has become available for a variety of organisms that offer distinct advantages over mice and rats for ease in conducting high-throughput chemical or mutagenesis screens. It is thus timely to examine the physiology and pathophysiology of the kidney or kidney equivalents in these organisms to evaluate their relevance as models for human disease. In addition to organisms whose small size and accessibility facilitate large scale screening approaches, larger animals at the other end of the spectrum offer unique physiological advantages in both size equivalency to humans as well as, in some cases, physiological and pathophysiological responses that closely mimic those of humans. Here we review a selected number of non-rodent experimental models of kidney diseases, focusing on recent advances in the use of the worm Caenorhabditis elegans, the fruitfly Drosophila melanogaster, the zebrafish Danio rerio, the little skate Leucoraja erinacea, the MGH miniature swine, merino cross sheep, and the cow Bos taurus to study kidney disease.

Mammalian Target of Rapamycin Mediates Kidney Injury Molecule 1-Dependent Tubule Injury in a Surrogate Model

Kidney injury molecule 1 (KIM-1), an epithelial phagocytic receptor, is markedly upregulated in the proximal tubule in various forms of acute and chronic kidney injury in humans and many other species. Whereas acute expression of KIM-1 has adaptive anti-inflammatory effects, chronic expression may be maladaptive in mice. Here, we characterized the zebrafish Kim family, consisting of Kim-1, Kim-3, and Kim-4. Kim-1 was markedly upregulated in kidney after gentamicin-induced injury and had conserved phagocytic activity in zebrafish. Both constitutive and tamoxifen-induced expression of Kim-1 in zebrafish kidney tubules resulted in loss of the tubule brush border, reduced GFR, pericardial edema, and increased mortality. Kim-1-induced kidney injury was associated with reduction of growth of adult fish. Kim-1 expression led to activation of the mammalian target of rapamycin (mTOR) pathway, and inhibition of this pathway with rapamycin increased survival. mTOR pathway inhibition in KIM-1-overexpressing transgenic mice also significantly ameliorated serum creatinine level, proteinuria, tubular injury, and kidney inflammation. In conclusion, persistent Kim-1 expression results in chronic kidney damage in zebrafish through a mechanism involving mTOR. This observation predicted the role of the mTOR pathway and the therapeutic efficacy of mTOR-targeted agents in KIM-1-mediated kidney injury and fibrosis in mice, demonstrating the utility of the Kim-1 renal tubule zebrafish models.