Derivation of pancreatic acinar cell carcinoma cell line HS-1 as a patient-derived tumor organoid.

Acinar cell carcinoma (ACC) of the pancreas is a malignant tumor of the exocrine cell lineage with a poor prognosis. Due to its rare incidence and technical difficulties, few authentic human cell lines are currently available, hampering detailed investigations of ACC. Therefore, we applied the organoid culture technique to various types of specimens, such as bile, biopsy, and resected tumor, obtained from a single ACC patient. Despite the initial propagation, none of these organoids achieved long-term proliferation or tolerated cryopreservation, confirming the challenging nature of establishing ACC cell lines. Nevertheless, the biopsy-derived early passage organoid developed subcutaneous tumors in immunodeficient mice. The xenograft tumor histologically resembled the original tumor and gave rise to infinitely propagating organoids with solid features and high levels of trypsin secretion. Moreover, the organoid stained positive for carboxylic ester hydrolase, a specific ACC marker, but negative for the duct cell marker CD133 and the endocrine lineage marker synaptophysin. Hence, we concluded the derivation of a novel ACC cell line of the pure exocrine lineage, designated HS-1. Genomic analysis revealed extensive copy number alterations and mutations in EP400 in the original tumor, which were enriched in primary organoids. HS-1 displayed homozygous deletion of CDKN2A, which might underlie xenograft formation from organoids. Although resistant to standard cytotoxic agents, the cell line was highly sensitive to the proteasome inhibitor bortezomib, as revealed by an in vitro drug screen and in vivo validation. In summary, we document a novel ACC cell line, which could be useful for ACC studies in the future.

Use of DNA-alkylating pyrrole-imidazole polyamides for anti-cancer drug sensitivity screening in pancreatic ductal adenocarcinoma.

BACKGROUND: Activating mutations of the KRAS occurs in >90% of pancreatic ductal adenocarcinoma (PDAC) cases. However, direct pharmacological targeting of the activated KRAS protein has been challenging. We previously reported that KR12, a DNA-alkylating pyrrole-imidazole polyamide designed to recognize the KRAS G12D/V mutation, showed an anti-tumor effect in colorectal cancer. In this study, we evaluated the anti-tumor effect of KR12 in PDAC. METHODS: KR12 was synthesized by an automated peptide synthesizer PSSM-8 and tested for anti-tumor effect in PDAC mouse models. RESULT: KR12 inhibited tumor growth in a spontaneous PDAC mouse model, although the anti-tumor activity appeared to be limited in a human PDAC xenograft model. We developed a pyrrole-imidazole polyamide screening process based on the hypothesis that genetic elements otherwise unaffected by KR12 could exert attenuating effects on KRAS-suppression-resistant PDAC. We identified RAD51 as a potential therapeutic target in human PDAC cells. A RAD51 inhibitor showed an inhibitory effect on cell growth and affected the cytotoxic activity of KR12 in PDAC cells. CONCLUSION: These data suggested that the simultaneous inhibition of RAD51 and mutant KRAS blockage would be an important therapeutic strategy for PDAC.

Efficacy of chemotherapy for patients with metastatic or recurrent pancreatic adenosquamous carcinoma: A multicenter retrospective analysis.

BACKGROUND/OBJECTIVES: Pancreatic adenosquamous carcinoma (PASC) is a rare variant of pancreatic ductal adenocarcinoma (PDAC). The usual treatment for metastatic or recurrent PASC is systemic chemotherapy in accordance with the PDAC treatment strategy. This study aimed to investigate the efficacy of chemotherapy, especially the benefit of recent combination therapies, in patients with metastatic or recurrent PASC. METHODS: We conducted a multicenter retrospective analysis of 116 patients with metastatic or recurrent PASC treated with first-line chemotherapy between April 2001 and December 2017 at 24 Japanese institutions. RESULTS: Combination chemotherapies included gemcitabine + nab-paclitaxel (GnP, n = 28), fluorouracil/leucovorin + irinotecan + oxaliplatin (FFX, n = 10), gemcitabine + S-1 (GS, n = 10), and others (n = 9). Monotherapies included gemcitabine (n = 51) and S-1 (n = 8). The median overall survival (OS) was 6.5, 7.3, and 4.3 months for the whole cohort, the combination therapy group, and the monotherapy group, respectively. Multivariate analysis indicated that combination therapy showed a better trend in OS than monotherapy (hazard ratio = 0.68; 95% confidence interval, 0.38-1.20). GnP or FFX were selected in 58.7% of patients after FFX was approved in Japan, and revealed a median OS, median progression-free survival, and objective response rate of 7.3 months, 2.8 months, and 26.9% in GnP and 7.2 months, 2.3 months, and 20.0% in FFX respectively. CONCLUSIONS: This study suggests that combination therapy may be more effective than monotherapy. GnP and FFX showed similar and clinically meaningful efficacy for patients with metastatic or recurrent PASC.

Endoscopic duodenal stent placement versus gastrojejunostomy for unresectable pancreatic cancer patients with duodenal stenosis before introduction of initial chemotherapy (GASPACHO study): a multicenter retrospective study.

BACKGROUND: Endoscopic duodenal stent placement is an alternative technique to gastrojejunostomy for gastric outlet obstruction due to pancreatic cancer. We compared the efficacy of endoscopic duodenal stent placement with that of gastrojejunostomy for treating patients with pancreatic cancer who are candidates for intensive combination chemotherapies as the first line of treatment. METHODS: This retrospective observational study included 100 patients from 18 institutions in Japan. Inclusion criteria were as follows: (1) cytologically or histologically confirmed adenocarcinoma of the pancreas, (2) good performance status, (3) gastric outlet obstruction scoring system score of 0-1 and (4) no history of treatment for pancreatic cancer. RESULTS: There was no significant difference in the background characteristics of patients in the endoscopic duodenal stent placement (n = 57) and gastrojejunostomy (n = 43) groups. The median overall survival in the endoscopic duodenal stent placement and gastrojejunostomy groups was 5.9 and 6.0 months, respectively. Clinical success was achieved in 93 cases; the median time to food intake resumption was significantly shorter in the endoscopic duodenal stent placement group (median: 3 days, n = 54) than in the gastrojejunostomy group (median: 5 days, n = 43). Chemotherapy was introduced in 63% of the patients in both groups after endoscopic duodenal stent placement or gastrojejunostomy. Chemotherapy was started earlier in the endoscopic duodenal stent placement group (median: 14 days) than in the gastrojejunostomy (median: 32 days) group. CONCLUSIONS: Endoscopic duodenal stent placement showed similar or better clinical outcomes than gastrojejunostomy. Thus, it might be a promising option in patients with good performance status.

Risk stratification and prognostic factors in patients with unresectable undifferentiated carcinoma of the pancreas.

BACKGROUND: Undifferentiated carcinoma (UC) of the pancreas has been considered a highly aggressive malignancy. However, only a few studies have systematically described the clinical course of UC patients. The aim of this study was to clarify the prognosis and construct a prognostic model for patients with unresectable UC. METHODS: This study was conducted at 17 institutions in Japan, and a total of 55 patients were analyzed. RESULTS: The median overall survival (OS) of patients with unresectable UC was 3.95 months. In the multivariate Cox proportional hazards (CPH) model, age ≥65 years, Eastern Cooperative Oncology Group performance status (ECOG PS) ≥2, and C-reactive protein (CRP) >10 mg/L were independent prognostic factors for OS (age ≥65 years: hazard ratio [HR], 2.732; 95% confidence interval [CI], 1.353-5.515; ECOG PS ≥ 2: HR, 7.866; 95% CI, 1.981-31.241; CRP >10 mg/L: HR, 1.956; 95% CI, 1.013-3.775). Based on the ß coefficients from the CPH model, the prognostic scores were defined as follows: age ≥65 years (3 points), ECOG PS ≥ 2 (6 points), and CRP >10 ml/L (2 points). The final prognostic model was the sum of the points. The derived prognostic model stratified patients into high-risk (score ≥4) and low-risk (score 0-3) groups, with significant differences in OS (1.45 vs. 8.19 months, respectively; p < 0.001). CONCLUSIONS: The prognostic model stratified patients into high-risk and low-risk groups. These findings suggest that this model can serve as a tool for patient information and decision-making with regard to the therapeutic strategy for UC.

Tenascin-C expression in renal biopsies from patients with tubulointerstitial nephritis and its relation to disease activity and prognosis.

Tenascin-C (TNC) is an extracellular matrix protein that is transiently expressed in close association with tissue remodeling in various organs. Expression of TNC in patients with tubulointerstitial nephritis (TIN) is not well-characterized. Using renal biopsy specimens from 25 patients with TIN and 8 patients with thin basement membrane disease (controls), we assessed immunohistochemical staining for TNC and investigated its relation with clinicopathologic data. TNC was undetectable in the controls, but TNC was observed in the interstitium of specimens from all patients with TIN, and strong TNC staining was detected within active tubulitis lesions. TNC was not principally expressed in glomeruli, and it was also absent from scar tissue. Comparison with Sirius red staining revealed that TNC was present where collagen fibers had not yet formed. The percent area of TNC within the interstitium (% TNC-positive area) showed a significant negative correlation with illness duration and significant positive correlations with the serum CRP level and eGFR aggravation, both of which reflect disease activity. On the other hand, no correlation was found between % TNC-positive area and eGFR recovery during 2 years of follow up. Examination of renal biopsy specimens from TIN patients revealed that TNC appears during the active stage of inflammation and then disappears with healing. This suggests that TNC expression reflects TIN disease activity, but not prognosis.

Vacuolated cardiomyocytes in human endomyocardial biopsy specimens.

We encountered an unfamiliar finding during electron microscopic examination of an endomyocardial biopsy obtained from a 55-year-old woman suffering from heart failure due to dilated phase hypertrophic cardiomyopathy. Many cardiomyocytes contained large vacuoles that were mainly empty except for small amounts of amorphous substrate. These were not autophagic vacuoles, as they lacked limiting membranes. Six years later, we encountered similar histological findings in three successive biopsies sourced from another hospital. They were obtained from a 77-year-old man with hypertrophic cardiomyopathy, a 28-year-old woman with endocardial fibrosis, and a 33-year-old man with dilated cardiomyopathy. This biopsy was the second for the endocardial fibrosis patient, and her first biopsy showed no vacuoles within cardiomyocytes. Close inspection of the procedures revealed that in all of these cases the fixed biopsy specimens were carried to the hospital from other institutes using a refrigerated courier service. We then fixed rat heart tissues, froze them once, and processed them for electron microscopy. In that experiment, we were able to reproduce the vacuolar cardiomyocytes, thereby demonstrating it to be a laboratory artifact. We therefore want to emphasize to physicians not to freeze biopsy specimens and not to use a refrigerated courier service for their transport. .

In situ nuclear DNA methylation in dilated cardiomyopathy: an endomyocardial biopsy study.

AIMS: Although distinct DNA methylation patterns have been reported, its localization and roles remain to be defined in heart failure. We investigated the cellular and subcellular localization of DNA methylation and its pathophysiological significance in human failing hearts. METHODS AND RESULTS: Using left ventricular (LV) endomyocardial biopsy specimens from 75 patients with dilated cardiomyopathy (DCM; age: 58 ± 14 years old, %female: 32%) and 20 patients without heart failure (controls; age: 56 ± 17 years old, %female: 45%), we performed immunohistochemistry and immunoelectron microscopy for methylated DNA, 5-methylcytosine (5-mC). We next investigated possible relations of the incidence of 5-mC-positive (%5-mC+ ) cardiomyocytes with clinicopathological parameters. Immunopositivity for 5-mC was detected in the cardiomyocytes and other cell types. The %5-mC+ cardiomyocytes was significantly greater in DCM hearts than in controls (57 ± 13% in DCM vs. 25 ± 12% in controls, P < 0.0001). The localization of 5-mC immunopositivity in cardiomyocyte nuclei coincided well with that of heterochromatin, as confirmed by immunoelectron microscopy. Substantial DNA methylation was also observed in interstitial non-cardiomyocytes, but the incidences did not differ between control and DCM hearts (39 ± 7.9% in DCM vs. 41 ± 10% in controls, P = 0.4099). In DCM patients, the %5-mC+ cardiomyocytes showed a significant inverse correlation with LV functional parameters such as heart rate (r = 0.2391, P = 0.0388), end-diastolic pressure (r = 0.2397, P = 0.0397), and ejection fraction (r = -0.2917, P = 0.0111) and a positive correlation with LV dilatation (volume index at diastole; r = 0.2442, P = 0.0347; and volume index at systole; r = 0.3136, P = 0.0062) and LV hypertrophy (mass index; r = 0.2287, P = 0.0484)-that is, LV remodelling parameters. No significant correlations between DNA methylation and the histological parameters of the biopsies, including cardiomyocyte hypertrophy, fibrosis, and inflammatory cell infiltration, were noted. CONCLUSIONS: The present study revealed increased nuclear DNA methylation in cardiomyocytes, but not other cell types, from DCM hearts, with predominant localization in the heterochromatin. Its significant relations with LV functional and remodelling parameters imply a pathophysiological significance of DNA methylation in heart failure.

Gemcitabine plus nab-paclitaxel for locally advanced or borderline resectable pancreatic cancer.

Overall survival in a phase III study for metastatic pancreatic cancer has significantly improved with gemcitabine (GEM) plus nab-paclitaxel. However, to date, there is limited data on the efficacy and safety of its use for patients with locally advanced (LA) or borderline resectable pancreatic cancer (BRPC). Here, we investigated the efficacy and safety of first-line GEM plus nab-paclitaxel for LA or BRPC. We retrospectively analysed consecutive patients with pathologically confirmed, untreated LA or BRPC who started receiving first-line GEM plus nab-paclitaxel. A total of 30 patients (LA, n = 22; BRPC, n = 8) were analysed. Twelve patients (40%) without distant metastasis received additional chemoradiotherapy using S-1. Laparotomy was performed on 8 patients and 6 (20%; LA, n = 3; BR, n = 3) achieved R0 resection. Objective response rate was 44.8%. For all patients, median progression-free survival and overall survival were 14.8 and 29.9 months, respectively. Median overall survival for LA was 24.1 months with a 2-year survival rate of 50.8%. The most frequently observed grade 3 or 4 toxicities were neutropenia (73%) and biliary infection (13%). First-line GEM plus nab-paclitaxel was well-tolerated and feasible with an encouraging survival for LA or BRPC.

Possible mechanism for disposal of degenerative cardiomyocytes in human failing hearts: phagocytosis by a neighbour.

The index case was a 51-year-old woman suffering from doxorubicin cardiomyopathy. In her endomyocardial biopsy specimen, we observed under electron microscopy six scenes in which degenerative cardiomyocytes were engulfed by neighbouring cardiomyocytes. The enclosed cardiomyocytes appeared more degenerative than the enclosing ones in every pair: the myofibrils were more severely damaged. At more degenerative stages, some desmosomes of the intercalated discs on the enclosed cardiomyocyte had disappeared. The membranes between the cardiomyocytes were occasionally disrupted, and there appeared to be sharing of cellular contents between the cells. One pair of such a phagocytosis-like figure was observed in one case with 5-fluorouracil cardiomyopathy (a 68-year-old man) among eight other chemotherapy-induced cardiomyopathies but none among 30 non-drug-induced dilated cardiomyopathies. The findings suggest a mechanism for disposal of degenerative cardiomyocytes in human failing hearts: phagocytosis by a neighbour, although alternative interpretations remain (e.g. giant autophagic vacuoles or two cardiomyocytes with degenerative intercalated discs).

Anti-apoptosis in nonmyocytes and pro-autophagy in cardiomyocytes: two strategies against postinfarction heart failure through regulation of cell death/degeneration.

Anti-apoptotic therapy for cardiomyocytes could be an effective strategy for preventing or treating heart failure. Notably, however, morphological evidence definitively demonstrating cardiomyocyte apoptosis has been very rare in actual heart diseases such as acute myocardial infarction and heart failure. By contrast, within the postinfarction heart, interstitial noncardiomyocytes such as granulation tissue cells do die via apoptosis to form scar tissue. Blockade of this apoptosis improves survival and mitigates ventricular remodeling and dysfunction during the chronic stage. Possible mechanisms to explain this benefit might be preservation of infarcted wall thickness and preservation of myofibroblasts, which could promote infarct shrinkage; both would reduce wall stress through Laplace's law. On the other hand, autophagy is an intracellular degradation mechanism that compensates for energy insufficiency by digesting and recycling intracellular components, and is often observed in cardiomyocytes within failing hearts with various origins including postinfarction. Starvation strongly induces and activates autophagic degeneration within cardiomyocytes. When that activation is inhibited, the starved animals suffer from heart failure. Promoting autophagy through caloric restriction or several reagents not only reduces the acute infarct size but also mitigates postinfarction cardiac remodeling and dysfunction during chronic stages. Moreover, augmenting autophagy by the treatment with resveratrol or exercise can bring about reverse remodeling in failing hearts with a large old myocardial infarction. In conclusion, we propose two strategies for managing postinfarction heart failure through control of cell death/degeneration: (1) anti-apoptosis in granulation tissue noncardiomyocytes; and (2) pro-autophagy in salvaged cardiomyocytes.

Ultrastructural Alteration of Pulmonary Capillary Endothelial Glycocalyx During Endotoxemia.

BACKGROUND: The most recent diagnostic criteria for sepsis include organ failure. Microvascular endothelial injury is believed to lead to the multiple organ failure seen in sepsis, although the precise mechanism is still controversial. ARDS is the primary complication during the sequential development of multiple organ dysfunction in sepsis, and endothelial injury is deeply involved. Sugar-protein glycocalyx coats all healthy vascular endothelium, and its disruption is one factor believed to contribute to microvascular endothelial dysfunction during sepsis. The goal of this study was to observe the three-dimensional ultrastructural alterations in the pulmonary capillary endothelium, including the glycocalyx, during sepsis-induced pulmonary vasculitis. METHODS: This study investigated the three-dimensional ultrastructure of pulmonary vascular endothelial glycocalyx in a mouse lipopolysaccharide-induced endotoxemia model. Lungs were fixed with lanthanum-containing alkaline fixative to preserve the glycocalyx. RESULTS: On both scanning and transmission electron microscopic imaging, the capillary endothelial glycocalyx appeared as a moss-like structure entirely covering the endothelial cell surface in normal mice. In the septic lung following liposaccharide injection, however, this structure was severely disrupted; it appeared to be peeling away and coagulated. In addition, syndecan-1 levels were significantly reduced in the septic lung, and numerous spherical structures containing glycocalyx were observed on the endothelial surface. CONCLUSIONS: It appears that endothelial glycocalyx in the lung is markedly disrupted under experimental endotoxemia conditions. This finding supports the notion that disruption of the glycocalyx is causally related to the microvascular endothelial dysfunction that is characteristic of sepsis-induced ARDS.

Three-dimensional ultrastructure of capillary endothelial glycocalyx under normal and experimental endotoxemic conditions.

BACKGROUND: Sugar-protein glycocalyx coats healthy endothelium, but its ultrastructure is not well described. Our aim was to determine the three-dimensional ultrastructure of capillary endothelial glycocalyx in the heart, kidney, and liver, where capillaries are, respectively, continuous, fenestrated, and sinusoidal. METHODS: Tissue samples were processed with lanthanum-containing alkaline fixative, which preserves the structure of glycocalyx. RESULTS: Scanning and transmission electron microscopy revealed that the endothelial glycocalyx layer in continuous and fenestrated capillaries was substantially thicker than in sinusoids. In the heart, the endothelial glycocalyx presented as moss- or broccoli-like and covered the entire luminal endothelial cell surface. In the kidney, the glycocalyx appeared to nearly occlude the endothelial pores of the fenestrated capillaries and was also present on the surface of the renal podocytes. In sinusoids of the liver, glycocalyx covered not only the luminal side but also the opposite side, facing the space of Disse. In a mouse lipopolysaccharide-induced experimental endotoxemia model, the capillary endothelial glycocalyx was severely disrupted; that is, it appeared to be peeling off the cells and clumping. Serum concentrations of syndecan-1, a marker of glycocalyx damage, were significantly increased 24 h after administration of lipopolysaccharide. CONCLUSIONS: In the present study, we visualized the three-dimensional ultrastructure of endothelial glycocalyx in healthy continuous, fenestrated, and sinusoidal capillaries, and we also showed their disruption under experimental endotoxemic conditions. The latter may provide a morphological basis for the microvascular endothelial dysfunction associated with septic injury to organs.

Ultrastructural aspects of vacuolar degeneration of cardiomyocytes in human endomyocardial biopsies.

Vacuolar degeneration of cardiomyocytes is a histological finding commonly encountered during routine light microscopic examination of human endomyocardial biopsy specimens. The vacuoles appear as intracellular clear areas lacking myofibers. By itself, this finding has little diagnostic value, but may have important clinical implications when the vacuolar contents are of etiological significance (e.g., accumulation of abnormal metabolites), and the clinical importance is increased when the disease is treatable. Thanks to its great resolving power, electron microscopy can often reveal the contents of the vacuoles and lead to a correct diagnosis. It can be used to differentially diagnose lysosomal storage diseases such as Fabry, Danon, and Pompe disease, doxorubicin cardiomyopathy, mitochondrial cardiomyopathy, autophagic degeneration, and accumulation of subcellular organelles (mitochondria, lipofuscin, glycogen granules, endoplasmic reticulum, etc.) as a nonspecific finding in failing cardiomyocytes. Nonetheless, undiagnosed cases certainly remain. It is strongly recommended that small pieces of tissue samples be fixed for electron microscopy at every endomyocardial biopsy procedure, and electron microscopic examination should be performed when a marked vacuolar degeneration is found.

Phase II study of induction gemcitabine and S-1 followed by chemoradiotherapy and systemic chemotherapy using S-1 for locally advanced pancreatic cancer.

PURPOSE: S-1 has systemic activity for locally advanced pancreatic cancer (LAPC). Here, the efficacy and safety of induction gemcitabine (GEM) and S-1 (GS) followed by chemoradiotherapy (CRT) and systemic chemotherapy using S-1 for LAPC were assessed. METHODS: The treatment consisted of four cycles of induction GS (S-1 60, 80, or 100 mg/day based on body surface area for 14 days every 3 weeks plus GEM 1000 mg/m2 on days 8 and 15), followed by S-1 (80, 100, or 120 mg/day based on body surface area on days 1-14 and 22-35) and concurrent radiotherapy (50.4 Gy in 28 fractions). Maintenance chemotherapy with S-1 was started 1-4 weeks after CRT until disease progression or unacceptable toxicity was observed. The primary endpoint was 1-year survival. RESULTS: A total of 30 patients with LAPC were enrolled. The median survival and progression-free survival were 21.3 and 12.7 months, respectively. Overall survival rates at 1, 2, 3, and 4 years were 73.3, 36.7, 23.3, and 16.7%, respectively. The median survival of 23 patients who received CRT was 22.9 months, with a 3-year survival rate of 30.4%. The two most common grade 3 or 4 adverse events during induction GS were neutropenia (63.3%) and biliary tract infection (20%). Toxicities during CRT or maintenance chemotherapy were generally mild. CONCLUSIONS: This regimen was feasible and highly active resulting in encouraging survival in patients with LAPC. Further investigations are warranted to elucidate the effectiveness of this treatment strategy in future studies. Clinical trials information: UMIN000006332.

Mitochondrial deformity confined to a single cardiomyocyte in human endomyocardial biopsy specimens: Report of 4 cases.

During electron microscopic examination of 156 consecutive human endomyocardial biopsy specimens, we found marked mitochondrial deformity within a single cardiomyocyte in each of 4 specimens. The deformed mitochondria were unevenly distributed, but the deformities were confined to the one cardiomyocyte. Those affected cardiomyocytes were accompanied by nonspecific degenerative changes such as nuclear hypertrophy and/or rarefaction of the myofibrils. Mitochondria in all other cells within the specimens appeared normal. Such an abnormality has never been reported to date. Each of the four cases was diagnosed with a different ailment: post-myocarditis, dilated cardiomyopathy, amyloidosis, and tachycardia-induced heart failure. However, all four cases were accompanied by left ventricular systolic dysfunction at biopsy. The very limited mitochondrial deformation may thus reflect a type of degenerative change that accompanies heart failure. .

Intravenous Administration of Endothelial Colony-Forming Cells Overexpressing Integrin ß1 Augments Angiogenesis in Ischemic Legs.

When injected directly into ischemic tissue in patients with peripheral artery disease, the reparative capacity of endothelial progenitor cells (EPCs) appears to be limited by their poor survival. We, therefore, attempted to improve the survival of transplanted EPCs through intravenous injection and gene modification. We anticipated that overexpression of integrin ß1 will enable injected EPCs to home to ischemic tissue, which abundantly express extracellular matrix proteins, the ligands for integrins. In addition, integrin ß1 has an independent angiogenesis-stimulating function. Human endothelial colony-forming cells (ECFCs; late-outgrowth EPCs) were transduced using a lentiviral vector encoding integrin ß1 (ITGB1) or enhanced green fluorescent protein (GFP). We then locally or systemically injected phosphate-buffered saline or the genetically modified ECFCs (GFP-ECFCs or ITGB1-ECFCs; 1 × 10(5) cells each) into NOD/Shi-scid, IL-2Rγnull mice whose right femoral arteries had been occluded 24 hours earlier. Upregulation of extracellular matrix proteins, including fibronectin, was apparent in the ischemic legs. Four weeks later, blood perfusion of the ischemic limb was significantly augmented only in the ITGB1-ECFC group. Scanning electron microscopy of vascular casts revealed increases in the perfused blood vessels in the ischemic legs of mice in the ITGB1-ECFC group and significant increases in the density of both capillaries and arterioles. Transplanted ECFC-derived vessels accounted for 28% ± 4.2% of the vessels in the ITGB1-ECFC group, with no cell fusion. Intravenous administration of ECFCs engineered to home to ischemic tissue appears to efficiently mediate therapeutic angiogenesis in a mouse model of peripheral artery disease. Significance: The intravenous administration of endothelial colony-forming cells (ECFCs) genetically modified to overexpress integrin ß1 effectively stimulated angiogenesis in ischemic mouse hindlimbs. Transplanted ECFCs were observed in the ischemic leg tissue, even at the chronic stage. Moreover, the cells appeared functional, as evidenced by the improved blood flow. The cell type used (ECFCs), the route of administration (intravenous, not directly injected into the affected area), and the use of ligand-receptor interactions (extracellular matrix and integrins) for homing represent substantial advantages over previously reported cell therapies for the treatment of peripheral artery disease.

Autophagic adaptations in diabetic cardiomyopathy differ between type 1 and type 2 diabetes.

Little is known about the association between autophagy and diabetic cardiomyopathy. Also unknown are possible distinguishing features of cardiac autophagy in type 1 and type 2 diabetes. In hearts from streptozotocin-induced type 1 diabetic mice, diastolic function was impaired, though autophagic activity was significantly increased, as evidenced by increases in microtubule-associated protein 1 light chain 3/LC3 and LC3-II/-I ratios, SQSTM1/p62 (sequestosome 1) and CTSD (cathepsin D), and by the abundance of autophagic vacuoles and lysosomes detected electron-microscopically. AMP-activated protein kinase (AMPK) was activated and ATP content was reduced in type 1 diabetic hearts. Treatment with chloroquine, an autophagy inhibitor, worsened cardiac performance in type 1 diabetes. In addition, hearts from db/db type 2 diabetic model mice exhibited poorer diastolic function than control hearts from db/+ mice. However, levels of LC3-II, SQSTM1 and phosphorylated MTOR (mechanistic target of rapamycin) were increased, but CTSD was decreased and very few lysosomes were detected ultrastructurally, despite the abundance of autophagic vacuoles. AMPK activity was suppressed and ATP content was reduced in type 2 diabetic hearts. These findings suggest the autophagic process is suppressed at the final digestion step in type 2 diabetic hearts. Resveratrol, an autophagy enhancer, mitigated diastolic dysfunction, while chloroquine had the opposite effects in type 2 diabetic hearts. Autophagy in the heart is enhanced in type 1 diabetes, but is suppressed in type 2 diabetes. This difference provides important insight into the pathophysiology of diabetic cardiomyopathy, which is essential for the development of new treatment strategies.