Publications by authors named "Keith Kobylarz"

8 Publications

  • Page 1 of 1

Infantile Hemangioma Originates From A Dysregulated But Not Fully Transformed Multipotent Stem Cell.

Sci Rep 2016 10 27;6:35811. Epub 2016 Oct 27.

Department of Medicine, New York University School of Medicine, 550 First Avenue New York, NY 10016, USA.

Infantile hemangioma (IH) is the most common tumor of infancy. Its cellular origin and biological signals for uncontrolled growth are poorly understood, and specific pharmacological treatment is unavailable. To understand the process of hemangioma-genesis we characterized the progenitor hemangioma-derived stem cell (HemSC) and its lineage and non-lineage derivatives. For this purpose we performed a high-throughput (HT) phenotypic and gene expression analysis of HemSCs, and analyzed HemSC-derived tumorspheres. We found that IH is characterized by high expression of genes involved in vasculogenesis, angiogenesis, tumorigenesis and associated signaling pathways. These results show that IH derives from a dysregulated stem cell that remains in an immature, arrested stage of development. The potential biomarkers we identified can afford the development of diagnostic tools and precision-medicine therapies to "rewire" or redirect cellular transitions at an early stage, such as signaling pathways or immune response modifiers.
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http://dx.doi.org/10.1038/srep35811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5081534PMC
October 2016

Tight regulation between cell survival and programmed cell death in GBM stem-like cells by EGFR/GSK3b/PP2A signaling.

J Neurooncol 2015 Jan 26;121(1):19-29. Epub 2014 Oct 26.

Laboratory for Translational Brain Tumor and Stem Cell Research, Department of Neurological Surgery, Weill Cornell Brain Tumor Center, Weill Cornell Medical College, 525 East 68th Street, Box 99, New York, NY, 10021, USA.

Malignant gliomas represent one of the most aggressive forms of cancer, displaying high mortality rates and limited treatment options. Specific subpopulations of cells residing in the tumor niche with stem-like characteristics have been postulated to initiate and maintain neoplasticity while resisting conventional therapies. The study presented here aims to define the role of glycogen synthase kinase 3 beta (GSK3b) in patient-derived glioblastoma (GBM) stem-like cell (GSC) proliferation, apoptosis and invasion. To evaluate the potential role of GSK3b in GBM, protein profiles from 68 GBM patients and 20 normal brain samples were analyzed for EGFR-mediated PI3kinase/Akt and GSK3b signaling molecules including protein phosphatase 2A (PP2A). To better understand the function of GSK3b in GBM, GSCs were isolated from GBM patient samples. Blocking GSK3b phosphorylation at Serine 9 attenuated cell proliferation while concomitantly stimulating apoptosis through activation of Caspase-3 in patient-derived GSCs. Increasing GSK3b protein content resulted in the inhibition of cell proliferation, colony formation and stimulated programmed cell death. Depleting GSK3b in GSCs down regulated PP2A. Furthermore, knocking down PP2A or blocking its activity by okadaic acid inactivated GSK3b by increasing GSK3b phosphorylation at Serine 9. Our data suggests that GSK3b may function as a regulator of apoptosis and tumorigenesis in GSCs. Therapeutic approaches targeting GSK3b in glioblastoma stem-like cells may be a useful addition to our current therapeutic armamentarium.
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http://dx.doi.org/10.1007/s11060-014-1602-3DOI Listing
January 2015

Sildenafil citrate improves erectile function after castration in a rat model.

BJU Int 2014 Apr 14;113(4):656-61. Epub 2013 Jun 14.

Department of Urology, Memorial Sloan-Kettering Cancer Center, New York, USA.

Take Home Message: The administration of phosphodiesterase 5 inhibitor commencing at the time of castration might preserve erectile function.

Objective: To determine if sildenafil citrate treatment could improve erectile function after castration. To determine if sildenafil citrate treatment reduces collagenisation and apoptosis in erectile tissue after castration.

Materials And Methods: In all, 60 Sprague-Dawley rats were studied; the rats were divided into the following groups: sham - no orchidectomy (S), control - orchidectomy only (O) and treatment - orchidectomy plus sildenafil treatment (V), with 10 rats per group. Erectile haemodynamics assessment was done at 7 days (S7, O7, V7) and at 28 days (S28, O28, V28) yielding a total of six groupings. Functional assessment measured the mean maximum intracavernosal pressure-mean arterial pressure (ICP/MAP) ratio. TUNEL assay was used to define apoptotic indices (AIs) and Masson's trichrome staining was used to evaluate smooth muscle-collagen (SM-C) ratios.

Results: The S28 group had the highest and the O7 group the lowest ICP/MAP ratio, at a mean (sd) of 70 (6)% and 36 (6)%, respectively. Both treatment groups, V7 [42 (12)%] and V28 [49 (13)%] showed statistically significant improvements over their corresponding control groups: O7 [36 (6)%] and O28 [37 (9)%] (P < 0.05). However, ICP/MAP values for V7 and V28 remained significantly below the S28 group (P < 0.001). There were no significant differences in ICP/MAP values between the 28-day and 7-day ICP/MAP ratios within each group (S, O, V). There were no significant differences in SM-C ratio between the O and V groups (O7 vs V7, P = 0.45; O28 vs V28, P = 0.16). There were no significant differences in AIs between the O and V groups (O7 vs V7, P = 0.54; O28 vs V28, P = 0.8).

Conclusions: Daily treatment with sildenafil improved erectile function in rats after castration. ICP/MAP ratios increased significantly in the treatment groups compared with the control groups with the greatest erectile function occurring 28 days from administration. In this series of experiments the improved erectile function recovery with sildenafil after surgical castration cannot be explained by smooth muscle protection and decreased collagenisation. The improved erectile function with sildenafil after surgical castration cannot be explained by reduced apoptosis in erectile tissue.
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http://dx.doi.org/10.1111/bju.12175DOI Listing
April 2014

p53-Associated Parkin-like cytoplasmic protein (Parc) short-interfering RNA (siRNA) alters p53 location and biology of Peyronie's disease fibroblasts.

BJU Int 2010 Dec;106(11):1706-13

Department of Urology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.

OBJECTIVE • To evaluate the impact of p53-associated Parkin-like cytoplasmic protein (Parc) short-interfering RNA (siRNA) on the location of p53 as well as the biology of Peyronie's disease (PD) plaque-derived fibroblasts after Parc knockdown. PATIENTS AND METHODS • Plaque tissue was excised from men with stable PD undergoing penile reconstructive surgery and used to produce cultured PD plaque-derived fibroblasts. • Immunofluorescence (IF) and reverse transcription-polymerase chain reaction (RT-PCR) were then used to define the location of p53 and Parc before and after siRNA. • Nuclear fractionation studies were used to assess the chronology of translocation of p53 from cytoplasm to nucleus on Parc knockdown. • The terminal transferase dUTP Nick end labelling (TUNEL) assay was used to assess the apoptotic indices of the PD fibroblasts after Parc knockdown. RESULTS • IF and PCR showed high cytoplasmic levels of p53 and Parc before siRNA. On IF, there was little or no p53 present within the nucleus before Parc knockdown. • After Parc siRNA, IF showed translocation of p53 to the fibroblast nucleus, while Parc levels dropped significantly, but what Parc remained was confined to the cytoplasm with none present in the nucleus. • Nuclear fractionation studies using RT-PCR confirmed this translocation phenomenon and showed the chronology of the event. All p53 had moved from the cytoplasm to the nucleus within 16 h of Parc siRNA. • On TUNEL assay, apoptotic indices increased dramatically after Parc siRNA. CONCLUSIONS • These data prove that Parc is a cytoplasmic anchor for p53 in PD plaque-derived fibroblasts and may be the primary cause of the stabilization and defunctionalization of p53 in these cells. • These findings support Parc as a novel target for PD pharmacotherapy, perhaps using human siRNA technologies once commercially available.
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http://dx.doi.org/10.1111/j.1464-410X.2010.09754.xDOI Listing
December 2010

The functional and structural consequences of cavernous nerve injury are ameliorated by sildenafil citrate.

J Sex Med 2008 May 4;5(5):1126-1136. Epub 2008 Mar 4.

Department of Urology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Introduction: Radical prostatectomy (RP) is associated with erectile dysfunction (ED). A single, placebo-controlled, human study has assessed the effects of regular sildenafil use after RP and demonstrated an increased chance of preservation of preoperative erectile function. Aim. This study was undertaken to define the effects of such a regimen in an animal model.

Methods: Using the cavernous nerve (CN) crush injury model, animals were divided into a number of groups: no CN injury (sham), bilateral CN injury exposed to either no sildenafil (control) or sildenafil at two doses (10 and 20 mg/kg) subcutaneously daily for three different durations (3, 10, 28 days).

Main Outcome Measures: At these time points, CN electrical stimulation was used to assess erectile function by mean intracavernosal pressure (ICP)/mean arterial pressure (MAP) ratio. For the structural analyses, whole rat penes were harvested. Staining for Masson's trichrome was utilized to calculate the smooth muscle-collagen ratio. Immunohistochemical antibody staining was performed for endothelial (CD31 and eNOS) and neural (GAP43, NGF, and nNOS) factors and immunoblotting was performed to analyze the AKT/eNOS pathway. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay was used for the assessment of apoptotic indices and the CN architecture was evaluated by transmission electron microscopy (TEM).

Results: Erectile function was improved with sildenafil in a time- and dose-dependent fashion with maximization of erectile function recovery occurring with daily 20 mg/kg at the 28-day time point. Sildenafil use resulted in smooth muscle-collagen ratio protection and CD31 and eNOS expression preservation. Sildenafil reduced apoptotic indices significantly compared with control. Animals exposed to sildenafil had increased phosphorylation of akt and eNOS. Tem demonstrated distinct differences in architecture between control and sildenafil groups toward an increased amount of myelinized nerve fibers.

Conclusions: Sildenafil use in the CN crush injury model preserves erectile function that appears to be mediated predominantly through preservation of smooth muscle content and endothelial function as well as through reduction in apoptosis.
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http://dx.doi.org/10.1111/j.1743-6109.2008.00794.xDOI Listing
May 2008

FK506 and erectile function preservation in the cavernous nerve injury model: optimal dosing and timing.

J Sex Med 2008 Jun 5;5(6):1334-44. Epub 2008 Mar 5.

Weill Medical College of Cornell University-Department of Urology, New York, NY, USA.

Introduction: The immunophilin-ligand FK506 has been shown to ameliorate erectile function and preserve cavernous nerve (CN) architecture in short-term-studies using rat models of CN injury.

Aim: The aim of this series was to ascertain the optimal dose and timing of FK506 administration in this animal model.

Methods: Rats underwent bilateral CN crush and were treated with FK506 at different time points. There were control (C) and sham groups for each time point. Based on preliminary experiments, the CN-crush rats had no treatment (C) or either FK506 1 mg/kg (BL) or 3.2 mg/kg (BH) for 3 days prior to and the day of CN crush (PRE), on the day of and for 3 days following CN crush (POST) and for 3 days pre-, on the day of, and 3 days post-CN crush (PP).

Main Outcome Measurements: All animals had measurement of intracavernosal pressure/mean arterial blood pressure (ICP/MAP) ratios at 28 days post-CN crush. Structural analysis was conducted in the POST groups. Penile tissue was assessed for apoptosis with terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay and immunohistochemically for neural factors (growth associated protein 43 [GAP43], nerve growth factor [NGF], and neural nitric oxide synthase [nNOS]). The CN architecture was examined by transmission electron microscopy (TEM).

Results: Sham animals had an ICP/MAP ratio of 70%. Only the BH-POST group revealed an improved ICP/MAP ratio compared with C (50 +/- 9% vs. 32 +/- 8%, P < 0.01). nNOS staining was significantly restored reaching sham levels in BL-POST and BH-POST groups vs. C (P < 0.05). NGF and GAP43 staining displayed no significant differences between C and treatment groups (P < 0.05). Apoptosis was significantly reduced in BL-POST and BH-POST groups compared with C (16 +/- 4%, 21 +/- 9%, and 63 +/- 7%, P < 0.001). TEM exhibited preservation of CN architecture for BH-POST compared with C.

Conclusion: These results suggest that short-term treatment with doses of FK506 higher than previously utilized preserves erectile function in the rat CN-injury model. Pretreatment appears to offer no advantage. However, FK506 administration just prior to CN injury and for a short-time post-injury achieves the best functional and structural preservation outcomes.
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http://dx.doi.org/10.1111/j.1743-6109.2008.00776.xDOI Listing
June 2008

The effect of hyperbaric oxygen therapy on erectile function recovery in a rat cavernous nerve injury model.

J Sex Med 2008 Mar 11;5(3):562-70. Epub 2008 Jan 11.

Department of Urology, Memorial Sloan Kettering Cancer Center, New York, NY 10021, USA.

Introduction: Cavernosal oxygenation appears to be important for preservation of erectile tissue health. Hyperbaric oxygen therapy (HBOT) has been shown to improve tissue oxygenation and has neuromodulatory effects.

Aim: This study was designed to define the effects of HBOT on erectile function (EF) and cavernosal tissue in the rat cavernous nerve (CN) injury model.

Methods: Four groups of Sprague-Dawley rats were studied: rats with bilateral CN crush, HBOT treated (Crush+/HBOT+); bilateral CN-crush/no HBOT (C+/H-); no crush/no HBOT (C-/H-); and no crush/HBOT (C-/H+). HBOT was delivered daily for 90 minutes at three atmospheres for 10 days commencing the day of CN crush.

Main Outcome Measures: Ten days after CN injury, the animals underwent CN stimulation measuring the maximal intracavernosal pressure/mean arterial pressure (ICP/MAP) ratios. Corporal tissue was harvested pre-sacrifice, and immunohistochemically stained for nerve growth factor (NGF), endothelial nitric oxide synthase (eNOS), and cluster of differentiation molecule (CD31). Histologic analysis was performed for Masson's trichrome to assess the smooth muscle-collagen ratio. Terminal deoxynucleotidyl transferase Biotin-dUTP Nick End Labeling assay was used to define apoptotic indices (AIs).

Results: The C+/H- group had significantly lower ICP/MAP ratios compared with C-/H- rats, (31% vs. 70%, P < 0.001). C+/H+ rats had significantly higher ICP/MAP ratio recovery compared with the C+/H- group (55% vs. 31%, P = 0.005). NGF and eNOS staining densities were higher in C+/H+ rats compared with C+/H- rats (P < 0.05 and P < 0.001, respectively). No difference was seen in CD31 expression. Staining density for MT displayed a trend toward higher smooth muscle preservation after HBOT. AIs were significantly increased by HBOT (P < 0.05).

Conclusion: HBOT following a CN injury improved EF preservation in this model, supporting the cavernosal oxygenation concept as protective mechanism for EF. The effects appear to be mediated via preservation of neurotrophic and endothelial factor expression.
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http://dx.doi.org/10.1111/j.1743-6109.2007.00727.xDOI Listing
March 2008

The impact of shock wave therapy at varied energy and dose levels on functional and structural changes in erectile tissue.

Eur Urol 2008 Mar 27;53(3):635-42. Epub 2007 Jun 27.

Department of Urology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.

Objectives: Only minimal literature exists on consequences of shock wave therapy (SWT) on erectile function in treatment of Peyronie's disease (PD). This study was undertaken to define SWT impact at varied energy/dose levels at different time points on functional and structural changes in erectile tissue.

Methods: In 45 rats 2000 shock waves (sw) at 2 BAR were applied to the penis weekly sorted by one, two, and three sessions (high-dose/energy level, HD-1, HD-2, HD-3). Each group was followed for 1, 7, or 28 d before measuring intracavernosal pressure (ICP) and mean arterial pressure (MAP). Fifteen control animals (C1, C7, C28) underwent anesthesia alone. Another 15 animals were exposed to three SWT sessions applying 1000 sw at 1 BAR and analyzed identically (low-dose/energy level, LD-3-1, -7, -28). Terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling assay was used to define the apoptotic index (AI) and Masson's trichrome (MT) staining was prepared to evaluate smooth muscle-to-collagen ratios.

Results: ICP/MAP ratios for all C groups displayed a mean of 64%. All SWT groups demonstrated significantly reduced ICP/MAP ratios compared to their corresponding C groups (p<0.05). The LD-3 groups showed a trend toward improved ICP/MAP ratios. LD-3-28 demonstrated significant recovery compared to HD-3-28 (55+/-8% vs. 41+/-10%, p=0.004), but remained reduced compared to C28 (63+/-5%, p=0.03). No statistical differences were seen for MT staining in SWT groups compared to C (p>0.05). AIs for the LD-3 groups were significantly lower compared to the HD-3 groups (p<0.001), but all AIs were significantly increased compared to C groups (p<0.01).

Conclusions: Overall, at both energy/dose levels, SWT resulted in a time- and treatment-dependent reduction of ICP/MAP ratios, which might be mediated partly through apoptosis and collagenization of corporal smooth muscle.
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http://dx.doi.org/10.1016/j.eururo.2007.06.032DOI Listing
March 2008