Bilateral Pedicled Scrotal Flaps as an Alternative to Skin Graft in Penile Shaft Defects Repair.

OBJECTIVE: To analyze surgical and functional outcomes of bilateral pedicled scrotal flaps for penile shaft reconstruction. MATERIALS AND METHODS: A retrospective analysis was performed on 22 patients who underwent penile shaft reconstruction with bilateral pedicled scrotal flaps between 2009 and 2017. Demographics, peri-operative data, and surgical complications were collected. Functional outcomes were analyzed using a questionnaire made of the erection hardness score, the patient and observer scar assessment scale, and a 10-point Likert scale measuring patients... satisfaction about their skin coloration, sensitivity, elasticity and thickness, penile size, scrotal volume, erection quality, penetration ability, pain, sexual satisfaction, body image, masculinity, self-esteem, and global satisfaction. RESULTS: Patients exhibited a wide range of indications, including buried penis (27.2%), or subcutaneous injections of foreign material (27.2%). Early complications were suture dehiscence (31.8%), infection (13.6%) and hematoma (4.6%), associated with 9.1% of surgical revisions. Late complications were skin retraction (27.3%), testicular ascension (22.7%), pyramidal shape (4.6%) or shortening (13.6%) of the penis, associated with 27.3% of surgical revisions. For the 12 patients who answered the questionnaire, median erection hardness score and patient and observer scar assessment scale score [IQR] were 3.5 out of 4 [2.5-4] and 11.5 out of 60 [9.5-22], respectively. The patients reported a positive impact of the surgery on their psychological condition, with a median score of global satisfaction of 8 [IQR 7.5-9.5]. CONCLUSION: Bilateral pedicled scrotal flaps seem.ßto be a safe alternative for shaft defects reconstruction despite a potential need of surgical revision, providing satisfactory functional outcomes.

Hypoxia and hypoxia-inducible factors promote the development of neointimal hyperplasia in arteriovenous fistula.

KEY POINTS: Patients with end-stage renal failure need arteriovenous fistulas (AVF) to undergo dialysis. However, AVFs present a high rate of failure as a result of excessive venous thickness. Excessive venous thickness may be a consequence of surgical dissection and change in oxygen concentration within the venous wall. We show that venous cells adapt their metabolism and growth depending on oxygen concentration, and drugs targeting the hypoxic response pathway modulate this response in vitro. We used the same drugs on a mouse model of AVF and show that direct or indirect inhibition of the hypoxia-inducible factors (HIFs) help decrease excessive venous thickness. Hypoxia and HIFs can be targets of therapeutic drugs to prevent excessive venous thickness in patients undergoing AVF surgical creation. ABSTRACT: Because the oxygen concentration changes in the venous wall, surrounding tissue and the blood during surgical creation of arteriovenous fistula (AVF), we hypothesized that hypoxia could contribute to AVF failure as a result of neointimal hyperplasia. We postulated that modulation of the hypoxia-inducible factors (HIF) with pharmacological compounds could promote AVF maturation. Fibroblasts [normal human fibroblasts (NHF)], smooth muscle cells [human umbilical vein smooth muscle cells (HUVSMC)] and endothelial cells [human umbilical vein endothelial cells (HUVEC)], representing the three layers of the venous wall, were tested in vitro for proliferation, cell death, metabolism, reactive oxygen species production and migration after silencing of HIF1/2-α or after treatment with deferioxamine (DFO), everolimus (Eve), metformin (Met), N-acetyl-l-cysteine (NAC) and topoisomerase I (TOPO), which modulate HIF-α stability or activity. Compounds that were considered to most probably modify intimal hyperplasia were applied locally to the vessels in a mouse model of aortocaval fistula. We showed, in vitro, that NHF and HUVSMC can adapt their metabolism and thus their growth depending on oxygen concentration, whereas HUVEC appears to be less flexible. siHIF1/2α, DFO, Eve, Met, NAC and TOPO can modulate metabolism and proliferation depending on the cell type and the oxygen concentration. In vivo, siHIF1/2α, Eve and TOPO decreased neointimal hyperplasia by 32%-50%, 7 days after treatment. Within the vascular wall, hypoxia and HIF-1/2 mediate early failure of AVF. Local delivery of drugs targeting HIF-1/2 could inhibit neointimal hyperplasia in a mouse model of AVF. Such compounds may be delivered during the surgical procedure for AVF creation to prevent early AVF failure.

Hepatic SRC-1 activity orchestrates transcriptional circuitries of amino acid pathways with potential relevance for human metabolic pathogenesis.

Disturbances in amino acid metabolism are increasingly recognized as being associated with, and serving as prognostic markers for chronic human diseases, such as cancer or type 2 diabetes. In the current study, a quantitative metabolomics profiling strategy revealed global impairment in amino acid metabolism in mice deleted for the transcriptional coactivator steroid receptor coactivator (SRC)-1. Aberrations were hepatic in origin, because selective reexpression of SRC-1 in the liver of SRC-1 null mice largely restored amino acids concentrations to normal levels. Cistromic analysis of SRC-1 binding sites in hepatic tissues confirmed a prominent influence of this coregulator on transcriptional programs regulating amino acid metabolism. More specifically, SRC-1 markedly impacted tyrosine levels and was found to regulate the transcriptional activity of the tyrosine aminotransferase (TAT) gene, which encodes the rate-limiting enzyme of tyrosine catabolism. Consequently, SRC-1 null mice displayed low TAT expression and presented with hypertyrosinemia and corneal alterations, 2 clinical features observed in the human syndrome of TAT deficiency. A heterozygous missense variant of SRC-1 (p.P1272S) that is known to alter its coactivation potential, was found in patients harboring idiopathic tyrosinemia-like disorders and may therefore represent one risk factor for their clinical symptoms. Hence, we reinforce the concept that SRC-1 is a central factor in the fine orchestration of multiple pathways of intermediary metabolism, suggesting it as a potential therapeutic target that may be exploitable in human metabolic diseases and cancer.

Increased gene copy number of VAMP7 disrupts human male urogenital development through altered estrogen action.

Despite the fact that genitourinary defects are among the most common birth defects in newborns, little is known about their etiology. Here we analyzed children born with congenital genitourinary tract masculinization disorders by array-comparative genomic hybridization, which revealed in 1.35% of cases the presence of de novo copy number gains at Xq28 encompassing the VAMP7 gene, which encodes a vesicle-trafficking protein that is part of the SNARE complex. Transgenic mice carrying a bacterial artificial chromosome encoding human VAMP7 mimicked the defective urogenital traits observed in boys with masculinization disorders such as cryptorchidism, urethral defects and hypospadias. Transgenic mice also exhibited reduced penile length, focal spermatogenic anomalies, diminished sperm motility and subfertility. VAMP7 colocalized with estrogen receptor α (ESR1) in the presence of its cognate ligand, 17ß-estradiol. Elevated levels of VAMP7 markedly intensified ESR1-potentiated transcriptional activity by increasing ESR1 protein cellular content upon ligand stimulation and upregulated the expression of estrogen-responsive genes including ATF3, CYR61 and CTGF, all of which have been implicated in human hypospadias. Hence, increased gene dosage of VAMP7, and thus higher expression levels of its protein product, enhances estrogen receptor action in male genitourinary tissues, affects the virilization of the reproductive tract and results in genitourinary birth defects in humans.

Increased expression of CYP24A1 correlates with advanced stages of prostate cancer and can cause resistance to vitamin D3-based therapies.

A major limitation of exogenous vitamin D3 administration for the treatment of prostate cancer is the marginal, if any, clinical efficacy. We dissected the basis for the resistance to the vitamin D3 antitumor properties and specifically examined the effect of its major catabolic enzyme, CYP24A1, in prostate cancer. Local CYP24A1 expression levels and the effect of selective modulation were analyzed using tissue microarrays from needle core biopsy specimens and xenograft-bearing mouse models. CYP24A1 mRNA was elevated in malignant human prostate tissues compared to benign lesions. High CYP24A1 protein levels were seen in poorly differentiated and highly advanced stages of prostate cancer and correlated with parallel increase in the tumor proliferation rate. The use of CYP24A1 RNAi enhanced the cytostatic effects of vitamin D3 in human prostate cancer cells. Remarkably, subcutaneous and orthotopic xenografts of prostate cancer cells harboring CYP24A1 shRNA resulted in a drastic reduction in tumor volume when mice were subjected to vitamin D3 supplementation. CYP24A1 may be a predictive marker of vitamin D3 clinical efficacy in patients with advanced prostate cancer. For those with up-regulated CYP24A1, combination therapy with RNAi targeting CYP24A1 could be considered to improve clinical responsiveness to vitamin D3.

Aberrations in pseudoautosomal regions (PARs) found in infertile men with Y-chromosome microdeletions.

CONTEXT: The pseudoautosomal regions (PARs) of the Y-chromosome undergo meiotic recombination with the X-chromosome. PAR mutations are associated with infertility and mental and stature disorders. OBJECTIVE: The aim of the study was to determine whether men with Y-chromosome microdeletions have structural defects in PARs. DESIGN AND PARTICIPANTS: Eighty-seven infertile men with Y-chromosome microdeletions and 35 controls were evaluated for chromosomal rearrangements using commercial or custom (X- and Y-chromosome) array comparative genomic hybridization or by quantitative PCR of selected PAR genes. Multisoftware-defined chromosomal gains or losses were validated by quantitative PCR and FISH. RESULTS: Array comparative genomic hybridization confirmed the AZF deletions identified by multiplex PCR. All men with Y-chromosome microdeletions and an abnormal karyotype displayed PAR abnormalities, as did 10% of men with Y-chromosome microdeletions and a normal karyotype. None of the control subjects or infertile men without Y-chromosome microdeletions had PAR duplications or deletions. SHOX aberrations occurred in 14 men (nine gains and five losses); four were short in stature (<10th percentile), and one was tall (>95th percentile). In contrast, the height of 23 men with Y-chromosome microdeletions and normal PARs was average at 176.8 cm (50th percentile). CONCLUSIONS: Y-chromosome microdeletions can include PAR defects causing genomic disorders such as SHOX, which may be transmitted to offspring. Previously unrecognized PAR gains and losses in men with Y-chromosome microdeletions may have consequences for offspring.

Identification of de novo copy number variants associated with human disorders of sexual development.

Disorders of sexual development (DSD), ranging in severity from genital abnormalities to complete sex reversal, are among the most common human birth defects with incidence rates reaching almost 3%. Although causative alterations in key genes controlling gonad development have been identified, the majority of DSD cases remain unexplained. To improve the diagnosis, we screened 116 children born with idiopathic DSD using a clinically validated array-based comparative genomic hybridization platform. 8951 controls without urogenital defects were used to compare with our cohort of affected patients. Clinically relevant imbalances were found in 21.5% of the analyzed patients. Most anomalies (74.2%) evaded detection by the routinely ordered karyotype and were scattered across the genome in gene-enriched subtelomeric loci. Among these defects, confirmed de novo duplication and deletion events were noted on 1p36.33, 9p24.3 and 19q12-q13.11 for ambiguous genitalia, 10p14 and Xq28 for cryptorchidism and 12p13 and 16p11.2 for hypospadias. These variants were significantly associated with genitourinary defects (P = 6.08×10(-12)). The causality of defects observed in 5p15.3, 9p24.3, 22q12.1 and Xq28 was supported by the presence of overlapping chromosomal rearrangements in several unrelated patients. In addition to known gonad determining genes including SRY and DMRT1, novel candidate genes such as FGFR2, KANK1, ADCY2 and ZEB2 were encompassed. The identification of risk germline rearrangements for urogenital birth defects may impact diagnosis and genetic counseling and contribute to the elucidation of the molecular mechanisms underlying the pathogenesis of human sexual development.

The coactivator SRC-1 is an essential coordinator of hepatic glucose production.

Gluconeogenesis makes a major contribution to hepatic glucose production, a process critical for survival in mammals. In this study, we identify the p160 family member, SRC-1, as a key coordinator of the hepatic gluconeogenic program in vivo. SRC-1-null mice displayed hypoglycemia secondary to a deficit in hepatic glucose production. Selective re-expression of SRC-1 in the liver restored blood glucose levels to a normal range. SRC-1 was found induced upon fasting to coordinate in a cell-autonomous manner, the gene expression of rate-limiting enzymes of the gluconeogenic pathway. At the molecular level, the main role of SRC-1 was to modulate the expression and the activity of C/EBPα through a feed-forward loop in which SRC-1 used C/EBPα to transactivate pyruvate carboxylase, a crucial gene for initiation of the gluconeogenic program. We propose that SRC-1 acts as a critical mediator of glucose homeostasis in the liver by adjusting the transcriptional activity of key genes involved in the hepatic glucose production machinery.

INSL3/RXFP2 signaling in testicular descent.

Mutations of the insulin-like peptide 3 (INSL3) hormone or its receptor, RXFP2, cause intraabdominal cryptorchidism in male mice. Specific RXFP2 expression in mouse gubernacula was detected at embryonic day 14.5 and markedly increased after birth in the developing cremaster muscle, as well as in the epididymis and testicular Leydig and germ cells. INSL3 treatment stimulated cell proliferation of embryonic gubernacular and Leydig cells, implicating active INSL3-mediated signaling. The transcription factor SOX9, a known male sex determination factor, upregulated the activity of the RXFP2 promoter. INSL3 is sufficient to direct the first transabdominal phase of testicular descent in the absence of hypothalamic-pituitary-gonadal axis signaling or Hoxa10, although these factors are important for inguinoscrotal testicular descent. Similarly, conditional ablation of the androgen receptor gene in gubernacular cells resulted in disruption of inguinoscrotal descent. We performed mutation screening of INSL3 and RXFP2 in human patients with cryptorchidism and control subjects from different populations in Europe and the USA. Several missense mutations were described in both the INSL3 and RXFP2 genes. A novel V39G INSL3 mutation in a patient with cryptorchidism was identified; however, the functional analysis of the mutant peptide did not reveal compromised function. In more than 2000 patients and controls analyzed to date, the T222P RXFP2 mutation is the only one strongly associated with the mutant phenotype. The T222P mutant receptor, when transfected into 293T cells, had severely decreased cell membrane expression, providing the basis for the functional deficiency of this mutation.

Oncogenic steroid receptor coactivator-3 is a key regulator of the white adipogenic program.

The white adipocyte is at the center of dysfunctional regulatory pathways in various pathophysiological processes, including obesity, diabetes, inflammation, and cancer. Here, we show that the oncogenic steroid receptor coactivator-3 (SRC-3) is a critical regulator of white adipocyte development. Indeed, in SRC-3(-/-) mouse embryonic fibroblasts, adipocyte differentiation was severely impaired, and reexpression of SRC-3 was able to restore it. The early stages of adipocyte differentiation are accompanied by an increase in nuclear levels of SRC-3, which accumulates to high levels specifically in the nucleus of differentiated fat cells. Moreover, SRC-3(-/-) animals showed reduced body weight and adipose tissue mass with a significant decrease of the expression of peroxisome proliferator-activated receptor gamma2 (PPARgamma2), a master gene required for adipogenesis. At the molecular level, SRC-3 acts synergistically with the transcription factor CAAT/enhancer-binding protein to control the gene expression of PPARgamma2. Collectively, these data suggest a crucial role for SRC-3 as an integrator of the complex transcriptional network controlling adipogenesis.

A tamoxifen-inducible chimeric Cre recombinase specifically effective in the fetal and adult mouse liver.

The spatiotemporal control of somatic mutagenesis in mice is considered a promising step to determine the function of a given gene product in a defined population of cells at any given time during animal life and also to generate better mouse models of human diseases. To introduce defined mutations in a temporally controlled manner in the liver, we established transgenic mice expressing a tamoxifen-inducible Cre recombinase under the control of the transthyretin promoter (TTR-Cre ind). The recombinase activity was examined on 2 different floxed alleles by crossing TTR-Cre ind mice with either the reporter strain ROSA 26 or with homozygous mice carrying floxed catalytic alpha2 subunit of the adenosine monophosphate (AMP)-activated protein kinase gene. By placing 2 mutated hormone-binding domains of murine estrogen receptor (Mer) at both termini of the Cre, we show that the fusion protein is active only on administration of the synthetic estrogen antagonist 4-hydroxytamoxifen (4-OHT) without any background in the absence of the inducing agent. The recombination is specific of the fetal and adult liver, and we show that the efficiency of recombination reached 80% to 100% after treatment with 4-OHT. In conclusion, TTR-Cre ind transgenic mice represent a valuable tool for temporally controlling the desired gene modifications in vivo in the fetal and adult liver. This would certainly help to understand the physiologic functions of genes in the liver, to create various mouse models mimicking human diseases, and to contribute to liver cancer-specific suicide gene therapy studies.