Severe left diaphragmatic hernia limits size of fetal left heart more than does right diaphragmatic hernia.

OBJECTIVES: To assess whether severity of congenital diaphragmatic hernia (CDH) correlates with the degree of left heart hypoplasia and left ventricle (LV) output, and to determine if factors leading to abnormal fetal hemodynamics, such as compression and reduced LV preload, contribute to left heart hypoplasia. METHODS: This was a retrospective cross-sectional study of fetuses at 16-37 weeks' gestation that were diagnosed with CDH between 2000 and 2010. Lung-to-head ratio (LHR), liver position and side of the hernia were determined from stored ultrasound images. CDH severity was dichotomized based on LHR and liver position. The dimensions of mitral (MV) and aortic (AV) valves and LV were measured, and right and left ventricular outputs were recorded. RESULTS: In total, 188 fetuses with CDH were included in the study, 171 with left CDH and 17 with right CDH. Fetuses with severe left CDH had a smaller MV (Z = -2.24 ± 1.3 vs -1.33 ± 1.08), AV (Z = -1.39 ± 1.21 vs -0.51 ± 1.05) and LV volume (Z = -4.23 ± -2.71 vs -2.08 ± 3.15) and had lower LV output (26 ± 10% vs 32 ± 10%) than those with mild CDH. MV and AV in fetuses with right CDH (MV, Z = -0.83 ± 1.19 and AV, Z = -0.71 ± 1.07) were larger than those in fetuses with left CDH, but LV outputs were similarly diminished, regardless of hernia side. Severe dextroposition and abnormal liver position were associated independently with smaller left heart, while LHR was not. CONCLUSION: The severity of left heart hypoplasia correlates with the severity of CDH. Altered fetal hemodynamics, leading to decreased LV output, occurs in both right- and left-sided CDH, but the additional compressive effect on the left heart is seen only when the hernia is left-sided. Improved knowledge of the physiology of this disease may lead to advances in therapy and better risk assessment for use in counseling affected families.

The new low-toxic histone deacetylase inhibitor S-(2) induces apoptosis in various acute myeloid leukaemia cells.

Histone deacetylase inhibitors (HDACi) induce tumour cell cycle arrest and/or apoptosis, and some of them are currently used in cancer therapy. Recently, we described a series of powerful HDACi characterized by a 1,4-benzodiazepine (BDZ) ring hybridized with a linear alkyl chain bearing a hydroxamate function as Zn(++)--chelating group. Here, we explored the anti-leukaemic properties of three novel hybrids, namely the chiral compounds (S)-2 and (R)-2, and their non-chiral analogue 4, which were first comparatively tested in promyelocytic NB4 cells. (S)-2 and partially 4--but not (R)-2--caused G0/G1 cell-cycle arrest by up-regulating cyclin G2 and p21 expression and down-regulating cyclin D2 expression, and also apoptosis as assessed by cell morphology and cytofluorimetric assay, histone H2AX phosphorylation and PARP cleavage. Notably, these events were partly prevented by an anti-oxidant. Moreover, novel HDACi prompted p53 and α-tubulin acetylation and, consistently, inhibited HDAC1 and 6 activity. The rank order of potency was (S)-2 > 4 > (R)-2, reflecting that of other biological assays and addressing (S)-2 as the most effective compound capable of triggering apoptosis in various acute myeloid leukaemia (AML) cell lines and blasts from patients with different AML subtypes. Importantly, (S)-2 was safe in mice (up to 150 mg/kg/week) as determined by liver, spleen, kidney and bone marrow histopathology; and displayed negligible affinity for peripheral/central BDZ-receptors. Overall, the BDZ-hydroxamate (S)-2 showed to be a low-toxic HDACi with powerful anti-proliferative and pro-apototic activities towards different cultured and primary AML cells, and therefore of clinical interest to support conventional anti-leukaemic therapy.

Upregulation of Bcl-2 through caspase-3 inhibition ameliorates ischemia/reperfusion injury in rat cardiac allografts.

BACKGROUND: Oxidative stress after ischemia/reperfusion of cardiac allografts leads to cytokine production. Bcl-2, an inhibitor of apoptosis, also has strong antioxidant properties. Caspase-3 is known to cleave bcl-2. This study tests the hypothesis that bcl-2 is downregulated while tumor necrosis factor-alpha (TNF-alpha) levels increase after cardiac transplantation. Furthermore, the use of caspase-3 inhibition was investigated as a strategy for preserving myocardial bcl-2 and mitochondrial cytochrome c after transplantation. METHODS AND RESULTS: PVG-to-ACI rat heterotopic cardiac transplantations were performed in 4 groups designed with 30 minutes' ischemia and 4 or 8 hours of reperfusion (n=4 per group). Treatment consisted of DEVD-CHO 500 microgram IP per animal to donor and recipient 2 hours before transplantation and 250 microgram IC into allograft. Controls were treated with saline. Grafts were analyzed by reverse transcription-polymerase chain reaction for bcl-2 mRNA, by ELISA for TNF-alpha, for myeloperoxidase activity, and by Western blot for cytochrome c. In untreated groups, bcl-2 mRNA decreased significantly over time, whereas TNF-alpha increased significantly at 4 hours (P=0.003) and returned to baseline after 8 hours' reperfusion (P=NS compared with normal hearts). Treatment with caspase-3 inhibitor showed significant upregulation of bcl-2 mRNA expression after 4 and 8 hours of reperfusion (P<0.001 versus control), with a concomitant decrease in TNF-alpha to baseline levels. Myeloperoxidase activity in all groups was no different from that of normal hearts. Mitochondrial cytochrome c release increased in both control and treatment groups. CONCLUSIONS: Bcl-2 is actively downregulated and TNF-alpha is upregulated in this model of cardiac allograft ischemia/reperfusion. Furthermore, the caspase-3 pathway is linked to this process, and blockade of caspase-3 can ameliorate reperfusion injury by upregulating bcl-2 and inhibiting TNF-alpha without affecting cytochrome c release.

L-arginine polymers inhibit the development of vein graft neointimal hyperplasia.

OBJECTIVE: We sought to determine whether L -arginine polymer treatment of vein grafts enhances vascular production of nitric oxide and inhibits the development of neointimal hyperplasia. METHODS: External jugular veins of New Zealand White rabbits (n = 42) were harvested; treated intraluminally for 15 minutes with phosphate-buffered saline solution or L -arginine polymer 5, 7, or 9 at either 10 or 100 micromol/L; and then grafted into the contralateral carotid artery. Rabbits were killed after 28 days, and 5-microm sections of vessels were stained with hematoxylin and scored for intima/media ratio by using computerized morphometric analysis. Separate veins were treated in a similar fashion with biotinylated polymers and phosphate-buffered saline solution to assess for translocation efficiencies. Finally, vein segments pretreated with either phosphate-buffered saline solution or L -arginine polymers were cultured in Dulbecco's modified Eagle's medium containing lipopolysaccharide (100 microg/mL) and interferon gamma (200 U/mL) for 48 hours before measuring nitric oxide levels by means of the Griess reaction. RESULTS: Biotinylated L -arginine polymers demonstrated a dose- and length-dependent uptake into intimal and medial cells of treated vessels. Nitric oxide levels were significantly higher in vein segments treated with 100 micromol/L of L -arginine polymer 9 compared with control segments. Finally, the intima/media ratio also reflected both length- and concentration-dependent inhibition of neointimal hyperplasia.intima/media ratio PBS R5 R7 R9 10 micromol/L 0.909 +/- 0.072 0.920 +/- 0.073 0.861 +/- 0.138 0.710 +/- 0.122 100 micromol/L 0.924 +/- 0.061 0.581 +/- 0.089* 0.529 +/- 0.093* PBS, Phosphate-buffered saline solution; R, L -arginine polymer. *P <.001 versus phosphate-buffered saline solution and L -arginine polymer 5 controls (Bonferroni-corrected value). CONCLUSIONS: Arginine polymers of sufficient length and concentration were effective in increasing nitric oxide levels and reducing neointimal hyperplasia in this vein graft model.

Techniques in orthotopic cardiac transplantation: a review.

Cardiac transplantation currently is the most effective therapy for end-stage heart failure. Since the origination of the standard biatrial technique, alternative methods such as the bicaval and "total" techniques have been devised with the hope of improving postoperative physiologic and clinical parameters. In general, the newer techniques are at least as effective as the original technique with respect to arrhythmia, valvular function, hemodynamics, exercise capacity, and survival, but whether any one technique offers clear benefits over another has been controversial. The bicaval technique is most commonly used today, and the general consensus is that this technique ultimately will demonstrate clinical superiority.

Ex vivo antisense oligonucleotides to proliferating cell nuclear antigen and Cdc2 kinase inhibit graft coronary artery disease.

BACKGROUND: The long-term success of cardiac transplantation is limited by graft coronary artery disease (GCAD). Antisense oligonucleotides (ASs) to proliferating cell nuclear antigen (PCNA) and Cdc2 kinase (Cdc2 k) can arrest cell cycle progression and inhibit neointimal hyperplasia. Transforming growth factor-ss(1) (TGF-ss(1)) has been implicated in vascular smooth muscle cell (VSMC) activation. The role of TGF-ss(1) in GCAD remains unclear. We hypothesized that ASs to PCNA and Cdc2 k would inhibit VSMC proliferation and GCAD. METHODS AND RESULTS: In vitro VSMC proliferation was determined after pretreatment with AS solution or medium alone followed by angiotensin II stimulation. PVG-to-ACI rat heterotopic cardiac transplantation procedures were performed after ex vivo pressure-mediated transfection of ASs to PCNA and Cdc2k or saline alone. At postoperative days 30, 60, and 90, allografts were assessed for GCAD, percent neointimal macrophages and VSMCs, and TGF-ss(1) activity. AS pretreatment significantly attenuated VSMC proliferation. At postoperative day 90, percent affected arteries, percent occlusion, and intima-media ratio demonstrated severe GCAD in saline-treated allografts, whereas these parameters were significantly lower in AS-treated allografts. Percent neointimal macrophages and VSMCs was reduced in AS-treated allografts. TGF-ss(1) activity was increased in saline compared with AS-treated allografts and nontransplanted heart controls. CONCLUSIONS: ASs to PCNA and Cdc2 k inhibit VSMC proliferation in vitro and reduce GCAD, percent neointimal VSMCs and macrophages, and TGF-ss(1) activity in vivo. TGF-ss(1) may play a "response to injury" role in the development of GCAD. The prevention of GCAD via AS inhibition of cell cycle regulatory genes before reperfusion may offer a useful clinical alternative to current therapeutic strategies.

Nuclear factor-kappaB transcription factor decoy treatment inhibits graft coronary artery disease after cardiac transplantation in rodents.

BACKGROUND: Nuclear factor-kappaB (NF-kappaB) is a transcription factor that upregulates adhesion molecules ICAM-1, VCAM-1, and ELAM-1. We hypothesized the use of ex vivo pressure-mediated delivery of transcription factor decoys (TFD) to NF-kappaB binding sites would decrease expression of adhesion molecules, and decrease reperfusion injury, acute rejection, and graft coronary artery disease (GCAD) in rat cardiac allografts. METHODS: Heterotopic heart transplants were performed on donor hearts treated with saline, 10 mg/kg LPS, 160 micromol/L NF-kappaB TFD, or 160 micromol/L scrambled sequence (NF-SC) TFD for 45 min at 78 psi (6 atm). Transfection efficiency was determined with FITC-labeled TFD. Reverse transcription-PCR and immunohistochemistry was used to analyze adhesion molecule mRNA and protein expression, respectively. Apoptosis was measured with DNA fragmentation analysis. Reperfusion injury was assessed with cardiac edema, neutrophil infiltration, and histology. Acute rejection was determined by daily palpation. Allografts were assessed at POD 90 for the development of GCAD by computer-assisted image analysis to determine intimal:medial ratio and myointimal proliferation. RESULTS: Hyperbaric pressure was an effective method of NF-kappaB TFD delivery (P<0.001 vs. controls). NF-kappaB TFD treatment led to decreased mRNA and protein expression of adhesion molecules. Treatment with NF-kappaB TFD led to a significant decrease in all reperfusion injury parameters compared to saline and NF-SC controls (P<0.01 vs. controls). Higher levels of apoptosis were seen in allografts treated with NF-kappaB TFD compared to control allografts. NF-kappaB TFD treatment prolonged allograft survival over saline and NF-SC controls (P<0.05). Myointimal proliferation and intimal:medial ratios in NF-kappaB TFD-treated allografts were significantly decreased compared to saline and NF-SC treatment (P<0.00001). CONCLUSIONS: Ex vivo pressure-mediated delivery of NF-kappaB TFD is an effective method to block adhesion molcule expression and reperfusion injury in the immediate posttransplant period. Further, NF-kappaB TFD treatment prolongs allograft survival and decreases GCAD.

Mechanical support for acutely failed heart or lung grafts.

Heart and lung allograft dysfunction continues to be a problem in thoracic transplantation. Although medical therapy is often sufficient to restore allograft function, occasionally more invasive means are required. Mechanical assist devices, inhaled nitric oxide (iNO), and extracorporeal membrane oxygenation (ECMO) have been used with a modest degree of success in cases of refractory heart, lung, and heart-lung allograft failure. Allograft failure secondary to pulmonary hypertension often responds to iNO concentrations between 5 and 70 ppm without major toxicity. More severe cases may require mechanical assist devices or ECMO and carry higher risks of complications such as bleeding, neurological injury, and death. Utilization of and weaning from these interventions require intensive monitoring. Randomized, prospective studies are not ethically feasible, but case reports and patient series indicate the usefulness of mechanical circulatory support, iNO, and ECMO. This review focuses on the indications, complications, and patient survival rates associated with these modalities.

Bone extracellular matrix induces homeobox proteins independent of androgens: possible mechanism for androgen-independent growth in human prostate cancer cells.

BACKGROUND: Differences in gene expression in prostate cells are believed to be secondary to epithelial-stromal interactions. We theorized that bone matrix may provide a fertile "soil" for prostate cancer by inducing androgen-dependent genes and allowing for androgen-independent growth. METHODS: Human prostate cancer cells (LNCaP) were grown under different conditions and analyzed for differential expression of mRNA. LNCaP cells were grown in the presence of 10 nM dihydrotestosterone (DHT), on extracellular matrix (ECM) derived from bone cells (without exogenous DHT), and on plastic culture dishes without exogenous DHT. A differential display of mRNA produced by LNCaP cells grown in the above conditions was then analyzed. RESULTS: Multiple unique transcripts were present in cells that were grown in the presence of DHT and on bone ECM (without exogenous DHT), but not on plastic culture dishes without exogenous DHT. Nine of these transcripts were then cloned and analyzed. Many (5/9) of these transcripts were found to contain multiple ATTA motifs in their corresponding 3'-untranslated regions. ATTA motifs have been shown to be homeobox protein-binding sites. Homeobox proteins and their target genes are thought to regulate cellular differentiation. Consistent with this, we demonstrated by reverse transcription polymerase chain reaction (PCR) that homeobox genes were differentially expressed in LNCaP cells when the cells were grown in the presence of DHT and on bone ECM (without exogenous DHT), but not on plastic culture dishes without exogenous DHT. Furthermore, we assayed LNCaP/fetal fibroblast chimeric tumors (n = 8) that were grown in male nude mice. Some of these tumors continued to grow in these mice despite treatment with surgical castration. In blinded studies, we were able to determine which tumor samples were androgen independent by their expression of homeobox genes. All samples that were androgen independent (n = 4) expressed the homeobox genes. Finally, gel retardation assay demonstrated that the homeobox proteins were able to bind to our cloned DNA sequences. Furthermore, footprinting analysis showed that the homeobox proteins bound to the ATTA motif in the 3'-region of our target DNA. CONCLUSIONS: Bone ECM, in the absence of DHT, has the ability to regulate androgen-responsive genes. Furthermore, many of these genes contain homeobox binding sites and the expression of homeobox genes may itself be regulated by bone ECM. If so, this may partially explain the clinical observation that bone provides a fertile "soil" for prostate cancer growth and metastasis.

Role of prostatic basal cells in the regulation and suppression of human prostate cancer cells.

Cytokeratin expression in normal and malignant prostatic tissue indicates a loss of basal epithelial cells in cancer. We investigated the ability of basal-like prostatic epithelial cells to inhibit the growth of prostatic cancer cells. Human prostate LNCaP cells were grown in medium with or without 10 nM dihydrotestosterone (DHT) on plastic culture dishes or on extracellular matrix derived from basal-like epithelial cells (primary cultures derived from normal peripheral zone of the prostate) that were grown with or without 10 nM DHT. Colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays were used to assess the growth of LNCaP cells. On plastic dishes, growth of LNCaP cells was increased 5-10% by the presence of DHT in the medium. On matrix derived from basal-like cells that were grown in the absence of DHT, growth of LNCaP cells with or without DHT was similar to that on plastic. However, on matrix derived from basal-like cells that were grown with DHT, growth of LNCaP cells was suppressed when compared to all other culture conditions (P < 0.01). To determine whether basal-like cells could alter the function of LNCaP cells, we measured prostate-specific antigen (PSA) mRNA expression with the use of comparative RT-PCR. We found a significant decrease in the mature PSA transcript in cells grown on matrix derived from basal-like cells that were grown with DHT. The expression of PSA transcript was not altered in LNCaP cells that were grown on matrix derived from basal-like cells that were grown in the absence of DHT. Furthermore, using differential display of mRNA, we demonstrated that there were induction and suppression of multiple unique transcripts in the LNCaP cells when grown on the various culture conditions. To determine a possible mechanism for these observations. We used a dot blot immunoassay for several known inhibitory factors. We determined that DHT can induce the basal-like cells to secrete transforming growth factor-beta (TGF-beta 1), and that TGF-beta 1 can inhibit the proliferation of LNCaP cells in a dose dependent manner. We conclude that basal-like epithelial cells, in the presence of DHT, secrete an extracellular matrix o matrix associated factor(s), e.g. TGF-beta 1, that suppresses proliferation and function of prostate cancer cells. Our data suggest that the disappearance of the basal cell layer may be a prerequisite for the progression of prostatic neoplasia.