Endothelial progenitor cells in morbid obesity.

BACKGROUND: The aim of this study was to assess the relationship among anthropometric indexes of adiposity (body mass index [BMI], waist circumference [WC]), endothelial progenitor cells (EPC) and carotid intima-media thickness (IMT) in patients with morbid obesity, and the effect of diabetes and weight loss. METHODS AND RESULTS: BMI, WC, IMT and circulating EPC (defined as CD34+/KDR+/CD45- cells) were assessed in 100 patients (37 with diabetes). Fifty patients underwent bariatric surgery, and in 48 of them a complete re-assessment after an average follow-up of 252±108 days was carried out. In 29 of them subcutaneous and visceral adipose tissue samples were obtained at the time of intervention and analyzed for the presence and number of EPC. EPC were directly correlated with weight, BMI, WC and insulin level, and inversely with mean IMT. All correlations were confined to non-diabetic patients. EPC were found in both subcutaneous and visceral adipose tissue specimens. Circulating EPC significantly decreased after weight loss (P=0.002). CONCLUSIONS: EPC are positively related to markers of adiposity in severe obesity, when not complicated by diabetes. Weight loss is associated with decrease in EPC level. EPC are inversely correlated with IMT, confirming their protective role also in severe obesity. Diabetes has a negative modulating action.

Effects of drospirenone-ethinylestradiol and/or metformin on CD4(+)CD28(null) T lymphocytes frequency in women with hyperinsulinemia having polycystic ovary syndrome: a randomized clinical trial.

OBJECTIVE: To evaluate the long-term effects of drospirenone (DRSP)/ethinylestradiol (EE) alone, metformin alone, and DRSP/EE-metformin on CD4(+)CD28(null) T lymphocytes frequency, a cardiovascular risk marker, in patients with hyperinsulinemic polycystic ovary syndrome (PCOS). DESIGN: Randomized clinical trial. INTERVENTIONS: Ninety three patients with hyperinsulinemic PCOS were age matched and body mass index matched and randomized to receive a 6 months daily treatment with DRSP (3 mg)/EE (0.03 mg), or metformin (1500 mg), or DRSP/EE combined with metformin. MAIN OUTCOME MEASURES: CD4(+)CD28(null) T-cell frequencies. RESULTS: The DRSP/EE and metformin groups did not show any significant change in the CD4(+)CD28(null) frequency compared to the baseline. Interestingly, a statistically significant decrease in CD4(+)CD28(null) frequency occurred after 6 months of DRSP/EE-metformin (median 3-1.5; P < .01). Of note, this statistically significant association was confirmed after adjusting for baseline values in DRSP/EE-metformin group by analysis of covariance (P < .05). CONCLUSIONS: In women with hyperinsulinemic PCOS, combined therapy with DRSP/EE and metformin may reduce cardiovascular risk.

Effect of exercise on circulating endothelial progenitor cells in microvascular angina.

BACKGROUND: Circulating endothelial progenitor cells (EPCs) might limit endothelial dysfunction in patients with microvascular angina (MVA). Endothelial colony-forming cells (ECFCs; displaying the CD34+/KDR+/CD45- phenotype) are currently regarded as true EPCs. The aim of this study was to evaluate exercise-induced ECFC mobilization and platelet reactivity in patients with MVA or with obstructive coronary artery disease (CAD). METHODS AND RESULTS: Exercise stress test (EST) was performed in 20 MVA patients, 20 CAD patients and 20 controls. Platelet reactivity was assessed before and after EST as formation of monocyte-platelet aggregates (MPAs) and CD41 platelet expression, without and with adenosine diphosphate (ADP) stimulation. ECFC number was measured before and 24h after EST. At rest, MPAs and CD41 platelet expression increased more with ADP in MVA patients (+71±11.0% and +37±7.5%, respectively), than in CAD patients (+37±8.6% and +19±4.5%, respectively) and controls (+29±3.5% and +21±3.1%, respectively; P<0.001 for both). At rest, ECFCs tended to be lower in CAD patients, compared to MVA patients and controls (4.1±5.0%, 7.2±6.0% and 7.3±7.0% cells/10(5), respectively; P=0.056). After EST, ECFCs increased less in MVA patients (+2.8±11) compared to CAD patients (+3.3±15; P<0.05) and controls (+7.4±24; P<0.01). CONCLUSIONS: In MVA patients, EST is able to blunt the peculiar increase of platelet reactivity to ADP present at rest; in contrast, no potential protective response of ECFCs to exercise was seen in these patients.

Differences in microparticle release in patients with acute coronary syndrome and stable angina.

BACKGROUND: Microparticles (MP) are vesicles released from activated or apoptotic cells. Endothelial MP (EMP) are derived from injured endothelium, platelet MP (PMP) from activated platelets, and Annexin V positive MP (AMP) from apoptotic endothelial cells. The aim was to assess the release of MP and its association with inflammation and atherosclerotic burden. METHODS AND RESULTS: AMP, EMP and PMP were measured on admission (Day 0) in 33 patients with stable angina (SA) and 43 patients with acute coronary syndrome (ACS) undergoing percutaneous coronary interventions (PCI). In SA, peripheral artery disease (PAD) was assessed by ultrasound examination. In 30 of the 76 patients (20 ACS and 10 SA), MP, high-sensitivity-C-reactive protein (hs-CRP), and troponin T (TnT) levels were also assessed 24h (Day 1) and 48 h (Day 2) after PCI. AMP, EMP, and PMP were higher in ACS than in SA (all P<0.01). In the SA group, AMP, PMP, and EMP were similar in patients with or without PAD. In the ACS group, AMP increased until Day 2 (P=0.001), while EMP and PMP peaked on Day 1 (P<0.01) then decreased to baseline values. Day 2 AMP correlated with Day 2 TnT levels (r=0.43, P=0.01) while Day 1 EMP and PMP correlated with Day 1 hs-CRP (r=0.37, P=0.04 and r=0.33, P=0.05; respectively). CONCLUSIONS: Higher MP levels were observed in ACS than in SA. Atherosclerotic burden did not affect MP levels in stable patients.

Intracoronary microparticles and microvascular obstruction in patients with ST elevation myocardial infarction undergoing primary percutaneous intervention.

AIMS: Microparticles (MP) are cell-derived fragments known to be increased in the blood of patients with acute coronary syndromes. We aimed to assess, in ST elevation myocardial infarction (STEMI), the systemic and local (in the culprit coronary artery) levels of platelet-derived MP (PMP, CD42+CD31+) and endothelial-derived MP (EMP, CD42-CD31+) and their relation to indexes of microvascular obstruction (MVO). METHODS AND RESULTS: In 78 STEMI patients undergoing successful primary percutaneous coronary intervention, blood samples were sequentially drawn from the aorta and the culprit coronary artery for cytofluorimetric MP detection. Thrombolysis in myocardial infarction (TIMI) flow, thrombus score (TS), corrected TIMI frame count (cTFC), myocardial blush grade (MBG), quantitative blush evaluator (QuBE) score, and 90 min ST resolution (ΣSTR) were calculated. Both PMP and EMP levels were significantly higher in the intracoronary than in the aortic blood samples. Intracoronary PMP and EMP levels were positively related to TS and cTFC and inversely related to MBG and QuBE. Aortic PMP (but not EMP) levels were related to TS and cTFC and, inversely, to QuBE. Intracoronary PMP were independently related to angiographic and electrocardiographic MVO in a multivariate model. CONCLUSION: The correlations of intracoronary EMP and of both systemic and intracoronary PMP levels with TS support the role of MP as markers of ongoing thrombosis. Moreover, the correlation of intracoronary MP with indexes of microvascular dysfunction suggests, for the first time, a possible direct role of MP in the pathogenesis of MVO.

Low-molecular-weight heparins induce decidual heparin-binding epidermal growth factor-like growth factor expression and promote survival of decidual cells undergoing apoptosis.

OBJECTIVE: To evaluate the effects of low-molecular-weight heparins (LMWHs) on decidual heparin-binding epidermal growth factor-like growth factor (HB-EGF) expression/secretion and on TNF-α-induced decidual apoptosis. DESIGN: Experimental study. SETTING: Department of Obstetrics and Gynecology, Università Cattolica del Sacro Cuore, Rome, Italy. PATIENT(S): Cultures of primary decidual cells isolated from human term placenta. INTERVENTION(S): The effects of LMWHs (tinzaparin and enoxaparin) on decidual HB-EGF expression and secretion were investigated by Western blot analysis and ELISA, respectively. TNF-α-induced decidual apoptosis was evaluated by annexin V staining, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assay, and caspase activities. MAIN OUTCOME MEASURE(S): Decidual HB-EGF expression/secretion and apoptotic rate induced by TNF-α were investigated. RESULT(S): Tinzaparin enhanced decidual HB-EGF expression and secretion. TNF-α reduced the number of viable cells by inducing apoptosis. Simultaneous addition of LMWHs (primarily tinzaparin) blocked the increase in annexin V- and TUNEL-positive cells and reduced the amount of caspase activities. CONCLUSION(S): Both LMWHs induced a significant increase in decidual HB-EGF expression/secretion and reduced TNF-α-induced decidual apoptosis. Tinzaparin demonstrated higher efficacy.

Are endothelial progenitor cells mobilized by myocardial ischemia or myocardial necrosis? A cardiac magnetic resonance study.

BACKGROUND: In ST-elevation myocardial infarction (STEMI) patients, the main stimuli involved in endothelial progenitor cells (EPCs) mobilization are not fully understood. We aimed to assess by cardiac magnetic resonance (CMR) whether the extent of ischemic myocardium (area at risk (AAR)) or of necrotic myocardium (infarct size (IS)) can be correlated to levels of circulating EPCs. METHODS: Peripheral EPCs were measured in fifteen STEMI patients at 24h after successful primary percutaneous coronary intervention (pPCI). Between two and four days after pPCI all patients underwent CMR assessment of myocardial AAR, IS, myocardial salvage (MS) and microvascular obstruction at late gadolinium enhancement CMR (LG-MVO). RESULTS: CD34+/KDR+, CD34+/KDR+/CD45dim, CD34+/KDR+/CD45-, EPCs were related to extent of AAR (rho=0.51, p=0.05; rho=0.55, p=0.03; rho=0.72, p=0.002, respectively), while no relationships were detected with IS, MS or LG-MVO. CONCLUSIONS: Our data show that EPCs were strongly correlated to extent of myocardial AAR, thus suggesting that progenitor cells mobilization in STEMI develops in response to myocardial ischemia and not to myocardial necrosis.

Pleiotropic beneficial effects of sonic hedgehog gene therapy in an experimental model of peripheral limb ischemia.

We have previously shown that the signaling pathway of the embryonic morphogen Sonic hedgehog (Shh) is recapitulated in the postnatal skeletal muscle in response to ischemia. We have also demonstrated that Shh is an indirect angiogenic agent upregulating various families of angiogenic growth factors and that Shh gene therapy improves angiogenesis and heart function in experimental models of myocardial ischemia. Based on these findings, we hypothesized that Shh gene therapy is beneficial in an experimental model of peripheral ischemia. We found that intramuscular (i.m.) treatment with a plasmid encoding the Shh human gene (phShh) increased blood flow, capillary density, and arteriole density in mice in which peripheral circulation of the hindlimb was disrupted by removal of the common femoral artery. Shh gene therapy also enhanced vasculogenesis, by increasing the number of circulating bone marrow (BM)-derived endothelial precursors and improving the contribution of these cells to the process of neovascularization. Finally, phShh treatment induced upregulation of prototypical angiogenic, arteriogenic, and vasculogenic factors, such as vascular endothelial growth factor (VEGF), angiopoietin 1 (Ang-1), and stromal cell-derived factor-1 (SDF-1α). These data suggest that Shh gene therapy merits further investigation for its ability to trigger the expression of potent trophic factors and stimulate pleiotropic aspects of neovascularization in the setting of ischemia.

Differential levels of circulating progenitor cells in acute coronary syndrome patients with a first event versus patients with recurring events.

OBJECTIVE: Pathophysiology of acute coronary syndromes in patients presenting with a first cardiac event (FCE) can be different from patients with a recurring cardiac event (RCE). We assessed inflammatory activation and circulating progenitor cells' (CPC) mobilisation in patients with a FCE versus those with RCE. METHODS: We recruited 41 patients: 18 with FCE and 23 with RCE. Peripheral blood samples were drawn at baseline and at 20 days to measure high sensitivity C-reactive protein (CRP) and to assess CD34+/133+ CPC and CD34+/KDR+ CPC by flow cytometry. RESULTS: CD34+/133+ cells (% number of cells per total number of cytometric events) were similar at baseline, being 0.25% (0.17-0.42%) in the FCE vs 0.23% (0.11-0.43%) in the RCE group, and increased at follow-up only in the FCE group to 0.41% (0.22-0.64%), while in the RCE group they were 0.27% (0.11-0.36%) (p=0.009 for the interaction, p=0.07 for the main effect of time). CD34+/KDR+ cells were similar at baseline in the two groups, did not significantly increase over time (p=0.2), and no differential effect of FCE vs RCE over time was seen (p=0.38). CRP levels, similar at baseline, were consistently reduced at 20 days after ACS (p=0.001), with no differential effect of FCE vs RCE pts (p=0.74). Variation from baseline to follow-up for both CD34+/133+ and CD34+/KDR+ did not correlate with either baseline CRP or delta CRP. CONCLUSIONS: Our data demonstrate a differential CPC mobilization behavior for FCE patients compared to RCE ones, independent of inflammatory activation.

Anti-tissue transglutaminase antibodies from celiac patients are responsible for trophoblast damage via apoptosis in vitro.

OBJECTIVES: The association between maternal celiac disease (CD) and both reduced fertility and increased risk of adverse pregnancy-related events has been long documented. However, no evidences are available regarding the pathogenic mechanisms of this link. The aim of this study was to determine whether anti-tissue transglutaminase (anti-tTG) antibodies are involved in the damage of trophoblastic cells in vitro. METHODS: Human primary trophoblastic cells, isolated from term placenta, were exposed to anti-tTG immunoglobulin G (IgG) antibodies, both commercially available and separated from sera of three untreated celiac women. The ability of anti-tTG antibodies to bind to trophoblastic cells, invasiveness of placental cells through a layer of extracellular matrix, and the activity of cellular matrix metalloprotease (MMP) and cellular apoptosis were evaluated, as indicators of trophoblast damage, by TdT-mediated dUTP digoxigenin nick end labeling (TUNEL) and annexin V expression. RESULTS: Anti-tTG IgG showed a specific dose- and time-dependent binding to human trophoblast. In addition, trophoblastic cells, after being exposed to anti-tTG IgG antibodies, both commercially available and separated from sera of celiac women, showed an impaired invasiveness, a decreased activity of cellular MMP, and a greater percentage of TUNEL positivity and annexin V positivity. CONCLUSIONS: We showed that the binding of anti-tTG antibodies to trophoblast might represent a key mechanism by which the embryo implantation and pregnancy outcome are impaired in untreated celiac pregnant women. Because healthy trophoblast development is essential for placental and fetal development, these data provide a novel mechanism for CD-induced infertility, early pregnancy loss, and intrauterine growth retardation.

Comparison of the effects of ramipril versus telmisartan on high-sensitivity C-reactive protein and endothelial progenitor cells after acute coronary syndrome.

To compare the anti-inflammatory and endothelial progenitor cell mobilizing effects of ramipril and telmisartan in patients presenting with acute coronary syndrome (ACS), 42 patients with ACS were randomized after successful percutaneous coronary intervention to ramipril 5 mg/day (22 patients) or telmisartan 80 mg/day (20 patients). Peripheral blood samples were drawn at baseline and at 20 days to measure high-sensitivity C-reactive protein and to assess 4 populations of progenitor cells by flow cytometry, namely CD34+/KDR+, CD34+/CD133+, CD34+/CD133+/CD45-, and CD34+/KDR+/CD45- cells. High-sensitivity C-reactive protein levels, similar in the 2 groups at baseline, were significantly more decreased by telmisartan than by ramipril at follow up (p = 0.013 for time-by-drug interaction). The main effect for time was also significant (p <0.001). CD34+/KDR+ and CD34+/CD133+ cells were similar at baseline and did not change over time (p = 0.2 and p = 0.1, respectively). In contrast, for CD34+/KDR+/CD45- and CD34+/CD133+/CD45- cells, a significant increase with time was seen (p = 0.02 and p = 0.002, respectively) and no differential effect of either drug was seen. In conclusion, telmisartan shows a more potent anti-inflammatory effect than ramipril after an ACS. The 2 drugs do not show a differential effect on endothelial progenitor cell mobilization.

Spontaneous myogenic differentiation of Flk-1-positive cells from adult pancreas and other nonmuscle tissues.

At the embryonic or fetal stages, autonomously myogenic cells (AMCs), i.e., cells able to spontaneously differentiate into skeletal myotubes, have been identified from several different sites other than skeletal muscle, including the vascular compartment. However, in the adult animal, AMCs from skeletal muscle-devoid tissues have been described in only two cases. One is represented by thymic myoid cells, a restricted population of committed myogenic progenitors of unknown derivation present in the thymic medulla; the other is represented by a small subset of adipose tissue-associated cells, which we recently identified. In the present study we report, for the first time, the presence of spontaneously differentiating myogenic precursors in the pancreas and in other skeletal muscle-devoid organs such as spleen and stomach, as well as in the periaortic tissue of adult mice. Immunomagnetic selection procedures indicate that AMCs derive from Flk-1(+) progenitors. Individual clones of myogenic cells from nonmuscle organs are morphologically and functionally indistinguishable from skeletal muscle-derived primary myoblasts. Moreover, they can be induced to proliferate in vitro and are able to participate in muscle regeneration in vivo. Thus, we provide evidence that fully competent myogenic progenitors can be derived from the Flk-1(+) compartment of several adult tissues that are embryologically unrelated to skeletal muscle.

Multiple interference of the human papillomavirus-16 E7 oncoprotein with the functional role of the metastasis suppressor Nm23-H1 protein.

High-risk human papillomaviruses (HPV) are linked to human cervical and other ano-genital cancers. Integration of the viral genome in the transformed epithelial cells is restricted to the coding regions for the E6 and E7 oncoproteins. Nevertheless, E7 plays the major role in cell transformation. We report a novel interaction between HPV-16 E7 and the Nm23-H1 and Nm23-H2 proteins identified in yeast by the two-hybrid system and confirmed by co-immunoprecipitation in the human keratinocyte HaCaT cell line. Expression of the E7 oncoprotein in HaCaT cells induces modified keratinocyte proliferation and differentiation patterns, and leads to down-modulation and functional inactivation of the metastasis suppressor Nm23-H1 protein. Both transcriptional down-regulation and protein degradation contribute to reduce Nm23-H1 intracellular content. Besides metastasis suppression, Nm23-H1 displays multiple functions in cell cycle regulation and differentiation, development, DNA regulation and caspase-independent apoptosis. As a consequence of Nm23-H1 inhibition, HPV-16 E7 expressing HaCaT cells, acquire invasiveness capabilities and resistance to granzyme A-induced apoptosis. We propose that impairment of the multifunctional role of Nm23-H1 is an important feature consistent with the complex strategy carried out by HPV-16 E7 to promote cell transformation and tumor progression.

Myogenic potential of adipose-tissue-derived cells.

Adipose-tissue-derived mesenchymal stem cells can be directed towards a myogenic phenotype in vitro by the addition of specific inductive media. However, the ability of these or other adipose-tissue-associated cells to respond to ;natural' myogenic cues such as a myogenic environment has never been investigated in detail. Here, we provide evidence that a restricted subpopulation of freshly harvested adipose-tissue-derived cells possesses an intrinsic myogenic potential and can spontaneously differentiate into skeletal muscle. Conversion of adipose-tissue-derived cells to a myogenic phenotype is enhanced by co-culture with primary myoblasts in the absence of cell contact and is maximal when the two cell types are co-cultured in the same plate. Conversely, in vitro expanded adipose-tissue-derived mesenchymal stem cells require direct contact with muscle cells to generate skeletal myotubes. Finally, we show that uncultured adipose-tissue-associated cells have a high regenerative capacity in vivo since they can be incorporated into muscle fibers following ischemia and can restore significantly dystrophin expression in mdx mice.

In vitro and in vivo tumor growth inhibition by a p16-mimicking peptide in p16INK4A-defective, pRb-positive human melanoma cells.

The cell cycle regulatory pathway responsible for the control of the late-G1 checkpoint is found recurrently altered in human malignant melanoma, often due to lack of functional p16 or pRb (pRb-1) proteins. Here we examined the ability of p16-derived peptides to mimic p16 function in two exemplary human melanoma cell lines: the p16-defective, pRb-positive A375M cells and p16-positive, pRb-defective A2058 cells. The synthetic p16-mimicking peptides strongly induced apoptosis in p16-, pRb+ A375M cells in vitro, while they had significantly less activity on p16+, pRb- A2058 cells. The most active p16-mimicking peptide, p16-AP9, also potently inhibited in vivo growth of the A375M melanoma. Treated tumors showed a threefold smaller volume (P < 0.025) and a significant reduction of the mitotic index and of PCNA expression. Growth of A2058 cells in vivo was not affected by treatment with the p16-mimicking peptide. Our results demonstrate that p16-mimicking peptides can induce apoptosis in vitro and that can inhibit tumor growth in vivo in p16-defective, pRb-expressing human melanoma cells, suggesting that p16-mimicking peptides can represent a promising tool for targeted therapy in selected cancer phenotypes.