Angela Criscimanna

Visceral Adiposity Index: A reliable indicator of visceral fat function associated with cardiometabolic risk

OBJECTIVE To individuate a novel sex-specific index, based on waist circumference, BMI, triglycerides, and HDL cholesterol, indirectly expressing visceral fat function. RESEARCH DESIGN AND METHODS Visceral adiposity index (VAI) was first modeled on 315 nonobese healthy subjects. Using two multiple logistic regression models, VAI was retrospectively validated in 1,498 primary care patients in comparison to classical cardio- and cerebrovascular risk factors. RESULTS All components of metabolic syndrome increased significantly across VAI quintiles. VAI was independently associated with both cardiovascular (odd ratio [OR] 2.45; 95% CI 1.52–3.95; P < 0.001) and cerebrovascular (1.63; 1.06–2.50; P = 0.025) events. VAI also showed significant inverse correlation with insulin sensitivity during euglycemic-hyperinsulinemic clamp in a subgroup of patients (Rs = −0.721; P < 0.001). By contrast, no correlations were found for waist circumference and BMI. CONCLUSIONS Our study suggests VAI is a valuable indicator of “visceral adipose function” and insulin sensitivity, and its increase is strongly associated with cardiometabolic risk.

Duct Cells Contribute to Regeneration of Endocrine and Acinar Cells Following Pancreatic Damage in Adult Mice

BACKGROUND & AIMS There have been conflicting results on a cell of origin in pancreatic regeneration. These discrepancies predominantly stem from lack of specific markers for the pancreatic precursors/stem cells, as well as differences in the targeted cells and severity of tissue injury in the experimental models so far proposed. We attempted to create a model that used diphtheria toxin receptor (DTR) to ablate specific cell populations, control the extent of injury, and avoid induction of the inflammatory response. METHODS To target specific types of pancreatic cells, we crossed R26DTR or R26DTR/lacZ mice with transgenic mice that express the Cre recombinase in the pancreas, under control of the Pdx1 (global pancreatic) or elastase (acinar-specific) promoters. RESULTS Exposure of PdxCre;R26DTR mice to diphtheria toxin resulted in extensive ablation of acinar and endocrine tissues but not ductal cells. Surviving cells within the ductal compartment contributed to regeneration of endocrine and acinar cells via recapitulation of the embryonic pancreatic developmental program. However, following selective ablation of acinar tissue in ElaCreERT2;R26DTR mice, regeneration likely occurred by reprogramming of ductal cells to acinar lineage. CONCLUSIONS In the pancreas of adult mice, epithelial cells within the ductal compartment contribute to regeneration of endocrine and acinar cells. The severity of injury determines the regenerative mechanisms and cell types that contribute to this process.

Multiple pluripotent stem cell markers in human anaplastic thyroid cancer: the putative upstream role of SOX2.

BACKGROUND Anaplastic thyroid carcinoma (ATC) is a rare and aggressive endocrine tumor with highly undifferentiated morphology. It has been suggested that cancer stem cells (CSCs) might play a central role in ATC. The objectives of this study were (i) to characterize CSCs from ex vivo ATC specimens by investigating the expression of several pluripotent stem cell markers, and (ii) to evaluate in vitro drug resistance modifications after specific CSC transcription factor switch-off. METHODS In ex vivo experiments, eight formalin-fixed, paraffin-embedded ATC specimens were analyzed by reverse-transcription and real-time quantitative PCR and immunohistochemistry. In in vitro experiments using ATC SW1736 cells, the expression levels of OCT-4, NANOG, and ABCG2 and the sensitivity to either cisplatin or doxorubicin were evaluated after silencing. RESULTS OCT-4, KLF4, and SOX2 transcription factors and C-KIT and THY-1 stem surface antigens showed variable up-regulation in all ATC cases. The SW1736 cell line was characterized by a high percentage of stem population (10.4±2.1% of cells were aldehyde dehydrogenase positive) and high expression of several CSC markers (SOX2, OCT4, NANOG, C-MYC, and SSEA4). SOX2 silencing down-regulated OCT-4, NANOG, and ABCG2. SOX2 silencing sensitized SW1736 cells, causing a significant cell death increase (1.8-fold) in comparison to control cells with 10 μM cisplatin (93.9±3.4% vs. 52.6±9.4%, p<0.01) and 2.7 fold with 0.5 μM doxorubicin (45.8±9.9% vs. 17.1±3.4% p<0.01). ABCG2 silencing caused increased cell death with both cisplatin (74.9±1.4%) and doxorubicin treatment (74.1±0.1%) vs. no-target-treated cells (respectively, 45.8±1.0% and 48.6±1.0%, p<0.001). CONCLUSIONS The characterization of CSCs in ATC through the analysis of multiple pluripotent stem cell markers might be useful in identifying cells with a stem-like phenotype capable of resisting conventional chemotherapy. In addition, our data demonstrate that SOX2 switch-off through ABCG2 transporter down-regulation has a major role in overcoming CSC chemotherapy resistance.

Recovery of Endogenous β-Cell Function in Nonhuman Primates After Chemical Diabetes Induction and Islet Transplantation

OBJECTIVE—To describe the ability of nonhuman primate endocrine pancreata to reestablish endogenous insulin production after chemical β-cell destruction. RESEARCH DESIGN AND METHODS—Eleven monkeys (Macaca fascicularis) were rendered diabetic with streptozotocin. Eight diabetic monkeys received intraportal porcine islet transplantation. RESULTS—Two monkeys transplanted after 75 days of type 1 diabetes showed recovery of endogenous C-peptide production a few weeks after transplantation, concomitant with graft failure. Histological analysis of the pancreas of these monkeys showed insulin-positive cells, single or in small aggregates, scattered in the pancreas and adjacent to ducts. Interestingly, numerous CK19+ cells costained with proinsulin and PDX-1 antibodies. Furthermore, the peculiar double phenotype glucagon-positive/GLUT2+ was observed. In these monkeys as well as in all others, the original islets showed no insulin staining. CONCLUSIONS—Our data provide evidence that, in nonhuman primates, the pancreas can reestablish endogenous insulin production after chemical β-cell destruction. This seems to be a nongeneralizable event with only 2 out of 11 monkeys recovering β-cell function. In these two monkeys, younger age and islet graft behavior might have played a role in triggering endogenous β-cell recovery.

PanIN-specific regulation of Wnt signaling by HIF2α during early pancreatic tumorigenesis

Hypoxia promotes angiogenesis, proliferation, invasion, and metastasis of pancreatic cancer. Essentially, all studies of the hypoxia pathway in pancreatic cancer research to date have focused on fully malignant tumors or cancer cell lines, but the potential role of hypoxia inducible factors (HIF) in the progression of premalignant lesions has not been critically examined. Here, we show that HIF2α is expressed early in pancreatic lesions both in human and in a mouse model of pancreatic cancer. HIF2α is a potent oncogenic stimulus, but its role in Kras-induced pancreatic neoplasia has not been discerned. We used the Ptf1aCre transgene to activate Kras(G12D) and delete Hif2α solely within the pancreas. Surprisingly, loss of Hif2α in this model led to markedly higher, rather than reduced, number of low-grade pancreatic intraepithelial neoplasia (mPanIN) lesions. These lesions, however, failed to progress to high-grade mPanINs, and displayed exclusive loss of β-catenin and SMAD4. The relationship among HIF2α, β-catenin, and Smad4 was further confirmed in vitro, where silencing of Hif2α resulted in reduced β-catenin and Smad4 transcript levels. Thus, with oncogenic Ras expressed in the pancreas, HIF2α modulates Wnt-signaling during mPanIN progression by maintaining appropriate levels of both Smad4 and β-catenin.

Specific transduction and labeling of pancreatic ducts by targeted recombinant viral infusion into mouse pancreatic ducts

Specific labeling of pancreatic ducts has proven to be quite difficult. Such labeling has been highly sought after because of the power it would confer to studies of pancreatic ductal carcinogenesis, as well as studies of the source of new insulin-producing β-cells. Cre-loxp recombination could, in theory, lineage-tag pancreatic ducts, but results have been conflicting, mainly due to low labeling efficiencies. Here, we achieved a high pancreatic duct labeling efficiency using a recombinant adeno-associated virus (rAAV) with a duct-specific sox9 promoter infused into the mouse common biliary/pancreatic duct. We saw rapid, diffuse duct-specific labeling, with 50 and 89% labeling in the pancreatic tail and head region, respectively. This highly specific labeling of ducts should greatly enhance our ability to study the role of pancreatic ducts in numerous aspects of pancreatic growth, development and function.

Valproic Acid Limits Pancreatic Recovery after Pancreatitis by Inhibiting Histone Deacetylases and Preventing Acinar Redifferentiation Programs

The mechanisms by which drugs induce pancreatitis are unknown. A definite cause of pancreatitis is due to the antiepileptic drug valproic acid (VPA). On the basis of three crucial observations-that VPA inhibits histone deacetylases (HDACs), HDACs mediate pancreas development, and aspects of pancreas development are recapitulated during recovery of the pancreas after injury-we hypothesized that VPA does not cause injury on its own, but it predisposes patients to pancreatitis by inhibiting HDACs and provoking an imbalance in pancreatic recovery. In an experimental model of pancreatic injury, we found that VPA delayed recovery of the pancreas and reduced acinar cell proliferation. In addition, pancreatic expression of class I HDACs (which are the primary VPA targets) increased in the midphase of pancreatic recovery. VPA administration inhibited pancreatic HDAC activity and led to the persistence of acinar-to-ductal metaplastic complexes, with prolonged Sox9 expression and sustained β-catenin nuclear activation, findings that characterize a delay in regenerative reprogramming. These effects were not observed with valpromide, an analog of VPA that lacks HDAC inhibition. This is the first report, to our knowledge, that VPA shifts the balance toward pancreatic injury and pancreatitis through HDAC inhibition. The work also identifies a new paradigm for therapies that could exploit epigenetic reprogramming to enhance pancreatic recovery and disorders of pancreatic injury.

Induction of mouse pancreatic ductal differentiation, an in vitro assay

Despite recent technical advances for studying lineage tracing and gene functions, our knowledge of pancreatic duct progenitor cells and mechanisms involved in their differentiation remains a huge void in our understanding of pancreatic development. A deeper insight into ductal differentiation is needed because ductal cells may harbor pancreatic stem/progenitor cells that could give rise to new islets. Also, since the most common pancreatic tumors form structures expressing ductal cell-specific markers, studies of ductal development may provide better markers for pancreatic tumor classification. One major longstanding problem in the study of pancreatic ductal differentiation has been the lack of an effective in vitro model. We thus wished to develop an in vitro system for the study of pancreatic duct development. In doing so, we have developed a specific culture condition to promote ductal differentiation of E11.5 pancreatic rudiments. Normally, pancreatic explants cultured in vitro develop to form endocrine, acinar, as well as ductal cells. Here, we report that addition of a combination of EGF, fibroblast growth factor-10, and platelet-derived growth factor-AA to the explant cultures promotes ductal differentiation, while preventing endocrine and acinar differentiation. This culture system for differentiation and enrichment of pancreatic ductal cells may allow identification of gene(s) involved in ductal development.

Identification of Newly Committed Pancreatic Cells in the Adult Mouse Pancreas

Multipotent epithelial cells with high Aldehyde dehydrogenase activity have been previously reported to exist in the adult pancreas. However, whether they represent true progenitor cells remains controversial. In this study, we isolated and characterized cells with ALDH activity in the adult mouse or human pancreas during physiological conditions or injury. We found that cells with ALDH activity are abundant in the mouse pancreas during early postnatal growth, pregnancy, and in mouse models of pancreatitis and type 1 diabetes (T1D). Importantly, a similar population of cells is found abundantly in healthy children, or in patients with pancreatitis or T1D. We further demonstrate that cells with ALDH activity can commit to either endocrine or acinar lineages, and can be divided into four sub-populations based on CD90 and Ecadherin expression. Finally, our in vitro and in vivo studies show that the progeny of ALDH1+/CD90−/Ecad− cells residing in the adult mouse pancreas have the ability to initiate Pancreatic and duodenal homeobox (Pdx1) expression for the first time. In summary, we provide evidence for the existence of a sortable population of multipotent non-epithelial cells in the adult pancreas that can commit to the pancreatic lineage following proliferation and mesenchymal to epithelial transition (MET).

The Enigma of β-Cell Regeneration in the Adult Pancreas: Self-Renewal Versus Neogenesis

The pancreas is constituted by two distinctly different tissues: the exocrine component, i.e., pancreatic acinar cells that secrete digestive enzymes; and the endocrine component, the islets of Langerhans, constituted by hormone secreting cells. In the islet, the ┙-cells produce glucagon; the ┚-cells, insulin; the ├-cells, somatostatin; ┛-cells, pancreatic polypeptide (Figure 1). Diabetes is caused either by an absolute (type 1) or relative (type 2) defect of insulin-producing ┚-cells in the pancreas. Therefore, regardless of the different pathogenesis, diabetes is the perfect candidate for cell replacement therapy. Currently the two available alternatives for ┚-cell replacement therapy are whole pancreas or isolated islet transplantation (Shapiro et al., 2000). However, these approaches are severely limited by a shortage of human organ donors and the need of lifelong immunosuppressive therapy. In the absence of other clearly suitable and renewable sources of surrogate ┚-cells, an alternative strategy to exogenous cell replacement therapy might be fostering endogenous ┚-cell regeneration. Therefore, knowledge of the mechanisms regulating ┚-cell plasticity in both embryonic and adult life, as well as in pathological conditions, is of particular interest. During pancreatic development, ┚-cells derive from a population of endocrine precursors arising from the pancreatic epithelium (Gittes, 2009). Activation of cell-specific transcription factors guides the initially multipotent progenitors and determines their differentiation into mature ┚-cells. The final size of the endocrine pancreas is limited by the size of the progenitor cell pool in the developing pancreatic bud (Stanger et al., 2007). After birth, most ┚-cells are considered quiescent, however, it has been shown that the ┚-cell mass can adaptively expand under some physiologic or pathologic circumstances, such as pregnancy and obesity, both in mammals and rodents (Bernard-Kargar & Ktorza, 2001). In mouse models there is also evidence that the pancreas preserves the ability to regenerate its ┚-cell mass in response to several non-physiological injuries, such as selective chemical destruction or surgical excision (Trucco, 2005; Thorel, 2010). Furthermore, in non-obese diabetic mice (NOD) it has been shown that recovery of sufficient endogenous insulin