Chiara Castellani

Widespread Increase in Myeloid Calcifying Cells Contributes to Ectopic Vascular Calcification in Type 2 Diabetes

Rationale: Acquisition of a procalcific phenotype by resident or circulating cells is important for calcification of atherosclerotic plaques, which is common in diabetes. Objective: We aim to identify and characterize circulating calcifying cells, and to delineate a pathophysiological role for these cells in type 2 diabetes. Methods and Results: We demonstrate for the first time that a distinct subpopulation of circulating cells expressing osteocalcin and bone alkaline phosphatase (OC+BAP+) has procalcific activity in vitro and in vivo. The study of naïve patients with chronic myeloid leukemia indicated that OC+BAP+ cells have a myeloid origin. Myeloid calcifying OC+BAP+ cells (MCCs) could be differentiated from peripheral blood mononuclear cells, and generation of MCCs was closely associated with expression of the osteogenic transcription factor Runx2. In gender-mismatched bone marrow–transplanted humans, circulating MCCs had a much longer half-life compared with OC−BAP− cells, suggesting they belong to a stable cell repertoire. The percentage of MCCs was higher in peripheral blood and bone marrow of type 2 diabetic patients compared with controls but was lowered toward normal levels by optimization of glycemic control. Furthermore, diabetic carotid endoarterectomy specimens showed higher degree of calcification and amounts of cells expressing OC and BAP in the &agr;-smooth muscle actin–negative areas surrounding calcified nodules, where CD68+ macrophages colocalize. High glucose increased calcification by MCCs in vitro, and hypoxia may regulate MCC generation in vitro and in vivo. Conclusions: These data identify a novel type of blood-derived procalcific cells potentially involved in atherosclerotic calcification of diabetic patients.

Stem cell compartmentalization in diabetes and high cardiovascular risk reveals the role of DPP-4 in diabetic stem cell mobilopathy

Bone marrow (BM) derived stem and progenitor cells contribute to cardiovascular homeostasis and are affected by cardiovascular risk factors. We devised a clinical data-driven approach to test candidate stem cell mobilizing mechanisms in pre-clinical models. We found that PB and BM CD34+ cell counts were directly correlated, and that most circulating CD34+ cells were viable, non-proliferating and derived from the BM. Thus, we analyzed PB and BM CD34+ cell levels as a two-compartment model in 72 patients with or without cardiovascular disease. Self-organizing maps showed that disturbed compartmentalization of CD34+ cells was associated with aging and cardiovascular risk factors especially diabetes. High activity of DPP-4, a regulator of the mobilizing chemokine SDF-1α, was associated with altered stem cell compartmentalization. For validation of these findings, we assessed the role of DPP-4 in the BM mobilization response of diabetic rats. Diabetes differentially affected DPP-4 activity in PB and BM and impaired stem/progenitor cell mobilization after ischemia or G-CSF administration. DPP-4 activity in the BM was required for the mobilizing effect of G-CSF, while in PB it blunted ischemia-induced mobilization. Indeed, DPP-4 deficiency restored ischemia (but not G-CSF)-induced stem cell mobilization and improved vascular recovery in diabetic animals. In conclusion, the analysis of stem cell compartmentalization in humans led us to discover mechanisms of BM unresponsiveness in diabetes determined by tissue-specific DPP-4 dysregulation.

Stem-cell therapy in an experimental model of pulmonary hypertension and right heart failure: Role of paracrine and neurohormonal milieu in the remodeling process

BACKGROUND In this study we investigated the effect of human amniotic fluid stem (hAFS) cells and rat adipose tissue stromal vascular fraction GFP-positive cell (rSVC-GFP) therapy and the contribution of the paracrine and neurohormonal milieu to cardiac and pulmonary vascular remodeling in a rat model of pulmonary hypertension (PH) and right heart failure (RHF). METHODS Sprague-Dawley rats were injected with monocrotaline (MCT). Four million hAFS or rSVC-GFP cells were injected via the tail vein 3 weeks after MCT. RHF was confirmed by RV hypertrophy/dilation and by brain natriuretic peptide (BNP) level. Cytokine profile was assessed by Multiplex array. Stem cell (SC) differentiation was studied by immunofluorescence. RESULTS MCT rats showed eccentric RV hypertrophy with increased RV dilation (measured as right ventricular mass/right ventricular volume [RVM/RVV]: MCT, 1.46 ± 0.12; control, 2.33 ± 0.24; p = 0.01), and increased RV hypertrophy (measured as LVM/RVM: MCT, 1.58 ± 0.06; control, 2.83 ± 0.1; p < 0.00001), increased BNP (MCT, 5.2 ± 1.2; control, 1.5 ± 0.1; p < 0.001) and both pro- and anti-inflammatory cytokines. SC produced a fall of BNP (hAFS, 2.1 ± 0.7; rSVC-GFP, 1.98 ± 1.3; p < 0.001) and pro-inflammatory cytokines. Positive RV remodeling with decreased RV dilation (RVM/RVV: hAFS, 1.87 ± 0.44; rSVC-GFP, 2.12 ± 0.24; p < 0.03 and p < 0.05 vs MCT) and regression of RV hypertrophy (LVM/RVM: hAFS, 2.06 ± 0.08; rSVC-GFP, 2.16 ± 0.08; p < 0.00001 vs MCT) was seen together with a decrease in medial wall thickness of pulmonary arterioles (hAFS, 35.33 ± 2.78%; rSVC-GFP, 37.15 ± 2.92%; p = 0.0001 vs MCT). CONCLUSIONS SC engrafted in the lung, heart and skeletal muscle modulated the pro- and anti-inflammatory cytokine milieu, and produced a positive neurohormonal response. This was accompanied by positive cardiac and pulmonary vascular remodeling, with formation mainly of new vascular cells.

Role of morphologic parameters on endomyocardial biopsy to detect sub-clinical antibody-mediated rejection in heart transplantation.

BACKGROUND The present study evaluated if morphologic parameters detect signs of early sub-clinical or latent stages of antibody-mediated rejection (AMR) and their correlation with C4d staining in cardiac transplants recipients. METHODS The study reviewed 1,270 endomyocardial biopsies (EMB) from 131 patients. Of these, 61 stained positive for C4d in the absence of acute cellular rejection >2R. Sixty-six EMB specimens negative for C4d were matched for pre-transplant diagnosis, time after transplantation, age, and acute cellular rejection (ACR) grading. Histopathologic evaluation and C4d staining were performed on formalin-fixed, paraffin-embedded sections using the C4d polyclonal antibody. RESULTS Of the 8 histologic characteristics evaluated, only endothelial swelling (78.7% sensitivity, 28.8% specificity; positive likelihood ratio, 1.10) and interstitial edema (77% sensitivity, 31.8% specificity; positive likelihood ratio, 1.13) could be considered fair predictors of C4d capillary positivity. The presence of mononuclear cells in capillaries in relation to C4d positivity showed 39.3% sensitivity and 68.2% specificity. Combining the parameters endothelial swelling and mononuclear cells in capillaries, sensitivity was 31.1% (95% confidence interval [CI] 19.9-44.3) and specificity was 71.2% (95 CI, 58.8-81.7), with a positive likelihood ratio of 1.08 (95% CI, 0.68-1.84). CONCLUSIONS Our results showed that histologic parameters did not always detect signs of early sub-clinical or latent stages of AMR. Combining the parameters of endothelial swelling and intracapillary mononuclear cells did not significantly improve the sensitivity or specificity. Screening recommendations should, therefore, be modified to include more sensitive tests such as C4d staining in the routine protocol to improve patient risk stratification.

Extracellular matrix graft for vascular reconstructive surgery: evidence of autologous regeneration of the neoaorta in a murine model

OBJECTIVES The study aimed to evaluate the efficacy of the porcine small intestine submucosa extracellular matrix (SIS-ECM) in a murine model, as a possible vascular patch for clinical use in reconstructive vascular and potentially cardiac surgery. METHODS Fifteen adult male Sprague Dawley rats and five green fluorescent protein (GFP) rats were enrolled in this study. The SIS-ECM graft (6 mm long, 4 mm wide) was implanted for patch plasty on the abdominal aorta of the animal, after excising part of its anterior wall. Histology and immunohistochemistry were used to evaluate the results at 15, 30, 90 and 180 days post-surgery. RESULTS Graft re-population was demonstrated 15 days after implantation. The luminal surface of the regenerating tissue was partially covered by endothelial cells, and intimal hyperplasia occurred in the central part of the graft. Complete re-endothelialization of the patch with smooth muscle cells colonizing the graft and acting as the neoaortic wall was observed after 30 days. Near complete absorption of the biomaterial was observed after 180 days. No inflammatory cell reaction occurred. All animals survived and no graft aneurysm was observed. CONCLUSIONS A SIS-ECM patch allowed the colonization of host endothelial and smooth muscle cells in the graft. This material may be an ideal substitute for reconstructive vascular surgery, and its use could be extended to surgical repair of cardiac defects.

Intravascular macrophages in cardiac allograft biopsies for diagnosis of early and late antibody-mediated rejection

BACKGROUND The aim of our study was to evaluate the role of intravascular macrophages in the diagnosis of early and late antibody-mediated rejection (AMR) on endomyocardial biopsies (EMBs). METHODS We reviewed 1,420 consecutive EMBs from 131 patients and selected 75 C4d+ EMBs. The C4d+ group was compared with a control group (66 patients) matched for age, gender, date of transplantation, follow-up, immunosuppressive regimen and primary heart disease. A total of 141 EMBs were evaluated. Immunoperoxidase staining for C4d and CD68 were performed. Post-transplant IgG anti-HLA reactivity was investigated by Luminex technology. Clinical data were also collected. Fourteen EMBs were available from 11 symptomatic AMR patients. RESULTS Of the 141 EMBs evaluated, 53 were positive for intravascular macrophages (CD68); among them, 32 were also positive for C4d (32 of 53, 60.4%). Of the 88 CD68- EMBs, 43 were also C4d+ (43 of 88, 48.9%). Of the 53 CD68+ EMBs, 30 EMBs were within the first year since transplantation (30 of 53, 57.8%), and among these 21 were also positive for C4d (21 of 30, 70.0%). In the late period, among the 23 CD68+ EMBs (23 of 53, 42.2%) 11 were also positive for C4d (11 of 23, 47.8%). In the early period, intravascular macrophages were more common in symptomatic (3 of 3, 100%) than asymptomatic (3 of 11, 27.3%) patients. Sensitivity and specificity of intravascular macrophages in predicting donor-specific antibodies (DSA) within the first year were 50.0% and 100.0%, respectively. CONCLUSIONS Intravascular macrophages predict C4d, DSA and symptoms early after transplantation; however, in the late period, they are unable to identify patients with circulating DSA, C4d and/or symptoms.

Pathological changes and myocardial remodelling related to the mode of shunting following surgical palliation for hypoplastic left heart syndrome

BACKGROUND The modification of placing the shunt from the right ventricle to the pulmonary arteries, also known as Sano procedure, has allegedly improved results over the short term in surgical palliation of hypoplastic left heart syndrome with the Norwood procedure. With this in mind, we reviewed autopsied specimens from neonates and children who did not survive after either a classic arterio-pulmonary shunt, or the modified procedure with the shunt placed from the right ventricle to the pulmonary arteries, so as to evaluate the pathological substrates of the remodelling of the systemic right ventricle, assessing any differences induced by the 2 techniques. METHODS We obtained the hearts from 11 patients with neonatal diagnosis of hypoplastic left heart syndrome who died after the first or second stages of the Norwood sequence of operations, comparing them with 6 normal hearts matched for age and weight. Macroscopic, microscopic and morphometric analysis were performed on each specimen, evaluating the diameter of the myocytes, extracellular matrix remodelling in terms of fibrosis and type of collagen, and vascularization in terms of capillary density. RESULTS Hypertrophy of the myocytes was significantly increased in the hearts from patients having either a classic arterio-pulmonary or the ventriculo-pulmonary modification of the shunt compared to controls (p < 0.05). Myocardial fibrosis was increased in those having a shunt placed from the right ventricle to the pulmonary arteries when compared to the other 2 groups. The ratio of collagen I to collagen III was similar in those undergoing a classic arterio-pulmonary shunt compared to controls (0.94), but was lower in those having a shunt placed from the right ventricle to the pulmonary arteries (0.61), with an increase in collagen type III. The density of capillaries was lower in those who had undergone a classic arterial shunt when compared to the others. CONCLUSION We have shown greater remodelling of the ventricular myocardial extracellular matrix in patients having a shunt from the right ventricle to the pulmonary arteries when compared to those having a classic arterio-pulmonary shunt, with this remodelling progressing even after the neonatal period. This may influence a later suboptimal ventricular performance.

Bone-marrow–derived CXCR4-positive tissue-committed stem cell recruitment in human right ventricular remodeling

The epicardium contributes to cardiac formation, particularly during embryogenesis. It remains to be seen if it is also involved in postnatal myocardial homeostasis. This study evaluates the topographic distribution of stem cells (c-Kit) and extracardiac progenitor cells (CXCR4+) and their contribution to ventricular remodeling in a model of pressure volume overload leading to right ventricle hypertrophy. Eleven specimens with hypoplastic left heart syndrome were evaluated and compared with 6 normal hearts from subjects matched for age and weight. All underwent Norwood procedure with the right ventricle becoming a systemic one, with pressure and volume overload leading to right ventricle remodeling. Transmural cardiac tissue samples from the right ventricle were analyzed by immunohistochemistry and morphometry. This is the first study to demonstrate that c-Kit-positive progenitor cells and tissue-committed stem cells (CXCR4+/CD45-) are higher in children with systemic right ventricle remodeling. We also show that the localization of cardiac progenitor and recruited CXCR4+ stem cells in the myocardium is site specific in hearts with right ventricle hypertrophy. These cells are mainly scattered in the interstitium of the epicardial layer. In contrast, myocyte proliferation is not a key process in right ventricular hypertrophy. Induced by the overexpression of SDF-1α by the myocardium, CXCR4 cell mobilization resembles SDF-1 homing factor distribution, showing transmural enhanced expression from the endocardium toward the epicardium. The study provides evidences of the site-specific epicardial localization of stem cells in a model of pressure/volume overload and suggests that the epicardium acts as a permissive niche in normal and pathologic conditions.

Coronary cardiac allograft vasculopathy versus native atherosclerosis: difficulties in classification

Cardiac allograft vasculopathy is regarded as a progressive and diffuse intimal hyperplastic lesion of arteries and veins that leads to insidious vessel narrowing and to allograft ischemic disease, such as acute myocardial infarction or sudden cardiac death. The coronary lesions in transplanted hearts are considered as a particular type of arteriosclerosis with many similarities but also significant differences compared to native coronary atherosclerosis. It is particularly difficult for pathologists to systematically classify the lesions and to elucidate their origins, since over time, the allograft immune responses cause vascular pathology characterized by not only the onset of de novo fibrocellular lesions but also remodeling of already-existing native atherosclerotic lesions in the donor heart. Intraplaque hemorrhages, which result from newly formed leaky microvessels, may cause rapid increase of stenosis and generate a substrate for plaque destabilization. Comparing cardiac allograft vasculopathy from explanted hearts at autopsy with native coronary atherosclerosis from hearts removed at transplantation has revealed that ongoing intraplaque hemorrhages are also an important feature of cardiac allograft vasculopathy and may be important factors in the rapid progression of cardiac allograft vasculopathy.

The role of macrophage-derived urokinase plasminogen activator in myocardial infarct repair Urokinase attenuates ventricular remodeling

Cardiac plasmin activity is increased following myocardial ischemia. To test the hypothesis that macrophage-derived uPA is a key mediator of repair following myocardial infarction, we performed myocardial infarction on mice with macrophage-specific over-expression of uPA (SR-uPA mice). SR-uPA(+/0) mice and wild-type littermates were sacrificed at 5 days or 4 weeks after infarction and cardiac content of macrophages, collagen, and myofibroblasts was quantified. Cardiac function and dimensions were assessed by echocardiography at baseline and at 4 weeks post-infarction. At 4 weeks after myocardial infarction, macrophage counts were increased in SR-uPA(+/0) mice in the infarct (13.1 vs. 4.9%, P<0.001) and distant uninfarcted regions (5.9 vs. 2.4%, P<0.001). Infarct scar was thicker in SR-uPA(+/0) mice (0.54+/-0.03 mm vs. 0.45+/-0.03 mm, P<0.05) and infarct cardiac collagen content was increased (72.4+/-3.3% vs. 63.0+/-3.6%, P<0.06). Functionally, these changes resulted in mildly improved fractional shortening in SR-uPA(+/0) mice compared to controls (24.6+/-1.68 vs. 19.8+/-1.3%, P=0.03). At 5 days after infarction there was increased collagen content in the scar without increases in macrophages or myofibroblasts. To understand the mechanisms by which macrophage-derived uPA increases collagen, cardiac fibroblasts were treated with macrophage-conditioned medium or plasmin and expression of ColIalpha1 measured by qPCR. Conditioned media from SR-uPA(+/0) or plasmin-treated non-transgenic macrophages but not plasmin alone increased collagen expression in isolated cardiac fibroblasts. We hypothesize that plasmin generation in the heart in response to injury may induce activation of macrophages to a profibrotic phenotype to allow rapid formation of collagenous scar.