Cystoscopic-guided laser ablation of intramural ectopic ureters in a female cat.

Case summary: An 8-month-old female spayed domestic shorthair cat was presented for chronic urinary incontinence (UI). Since being adopted 6 months earlier, the cat had a history of urine leakage during both activity and rest. Baseline blood work and urine culture showed no significant abnormalities and no evidence of a urinary tract infection. An abdominal CT with excretory urography followed by a focal urinary tract ultrasound revealed a suspected right intramural ectopic ureter (EU) and potential left EU. Cystoscopy confirmed bilateral intramural EUs. Cystoscopic-guided laser ablation (CLA) of both EUs was performed. The cat developed temporary urinary obstruction (UO) 36 h after the procedure, which was medically managed with prazosin and buprenorphine. Ultimately, the cat's urinary signs completely resolved with no UI recognized after the procedure and the cat has remained continent during 18 months of follow-up. Relevance and novel information: CLA of intramural EUs is routinely performed in dogs, but this technique has not been previously reported in cats with this condition. Although post-procedural urinary tract signs were initially present, the cat ultimately had an excellent outcome with resolution of UI after this procedure.

Accuracy of Crit-Line® monitors in predicting hematocrit and change in blood volume of dogs during hemodialysis.

BACKGROUND: Ultrafiltration (UF) is an extracorporeal technique for treating fluid overload and is monitored with noninvasive blood volume monitors. HYPOTHESIS/OBJECTIVES: The primary objective was to determine the accuracy of Crit-Line® III and IV noninvasive blood volume monitors to estimate canine packed cell volume (PCV) and changes in blood volume during UF. A secondary aim was to determine accuracy of targeted ultrafiltration rates (UFR) compared to calculated delivered UFR. METHODS: An ex vivo study with a single Phoenix® X36 platform and canine packed red blood cells (pRBC) was performed. Through dilution and UF, clinically applicable PCV values were obtained and compared to Crit-Line® hematocrit (Hct). Blood volume was constantly measured and compared to targeted UFR. Systematic and proportional bias were calculated using a Bayes method. RESULTS: Crit-Line® III and IV reported Hct was significantly lower than PCV (n = 140, median 26%, range, 8%-50%) when PCV was >25% and >30%, respectively. Crit-Line® III and IV calculated change in blood volume (ΔBV%) was significantly different from measured blood volume changes at ΔBV% ±20% and >-20%, respectively. Comparing targeted and delivered UFR (mL/h), less than targeted UFR was removed at UFR100 through UFR400 and UFR0 adding volume. CONCLUSIONS AND CLINICAL IMPORTANCE: Crit-Line® III and IV monitors provide accurate estimates of canine PCV and UF volume change within specific ranges and are useful in monitoring canine UF and hemodialysis. Veterinary hemodialysis services should evaluate individual machines for UFR inaccuracies, which can meaningfully affect small animals.

Ultrasonographic features of gallbladder wall thickening in dogs with hypoalbuminemia.

Hypoalbuminemia has been considered as a cause of gallbladder wall thickening in humans and dogs. Recent study revealed that gallbladder wall thickening in dogs with hypoalbuminemia may not be associated with serum albumin/plasma levels within 48 h of ultrasound study. However, gallbladder wall edema may change within 48 h, and the ultrasonographic features of gallbladder wall thickening in dogs with hypoalbuminemia have not been reported. The purpose of this study is to describe the relationship between serum albumin levels within 24 h of ultrasound and gallbladder wall thickening, and to describe the ultrasonographic features of thickened gallbladder walls in dogs with hypoalbuminemia. 37 hypoalbuminemic dogs with gallbladder ultrasound images were retrospectively included. Ultrasound studies were reviewed, and gallbladder wall thickness, layering appearance, echogenicity, echotexture, distribution, evidence of gallbladder mucocele, and presence of peritoneal effusion were recorded. Additionally, serum albumin levels within 24 h of ultrasound study and the administerd sedation were recorded. The prevalence of gallbladder wall thickening in dogs with hypoalbuminemia was 13.5%. The 3-layer appearance of the gallbladder wall was observed in 4 dogs, and a single-layer gallbladder wall thickening in one dog. Diffuse thickening was observed in all 5 dogs. The serum albumin level of dogs with gallbladder wall thickening was not different (p = 0.14) from dogs without thickening. Gallbladder wall thickening was not common, occurring only with mild hypoalbuminemia, and was commonly associated with a 3-layer appearance and considered as gallbladder wall subserosal edema. Causes other than hypoalbuminemia may be responsible for thickening of the gallbladder wall in dogs with hypoalbuminemia.

Iodinated non-ionic contrast medium does not inhibit Escherichia coli growth in ex vivo inoculated canine urine.

OBJECTIVE: To investigate the effect of iohexol on standardized quantitative urine culture results in dogs. The authors hypothesized that the presence of iohexol in inoculated urine samples would result in lower bacterial concentrations (CFU/mL) and, therefore, decrease culture sensitivity. SAMPLE: Urine samples were aseptically collected during cystoscopy from a single client-owned dog untreated with antimicrobials. PROCEDURES: An experimental controlled study. The urine sample was divided into 38 aliquots (0.5 mL each) that were used as negative controls or inoculated with an equal amount of Escherichia coli (105 CFU/mL). Different volumes (0.1 and 0.5 mL) of contrast or saline were added to the aliquots and quantitative culture results were compared. Two different incubation times between the preparation of aliquots and culture were evaluated (15 minutes and 24 hours). RESULTS: All aliquots from samples inoculated with E. coli (positive controls and iohexol-group) had the same reported quantitative result (104 CFU/mL). No growth was reported for the negative controls. Iohexol did not show any anti-E. coli properties in canine urine cultures for dilutions up to 1:2 contrast:urine and concentrations up to 120 mgI/mL. No difference was reported when iohexol was incubated with inoculated urine for 15 minutes or 24 hours. CLINICAL RELEVANCE: Based on the experimental in vitro conditions described, administration of iohexol before the collection of urine during urologic procedures does not negatively impact the isolation and growth of E. coli.

Concentrations and kinetics of renal biomarkers in dogs with gastric dilatation-volvulus with and without 24-h intravenous lidocaine.

Background: Gastric dilatation volvulus (GDV) can lead to organ failure including acute kidney injury (AKI). Due to its cytoprotective, antioxidant and anti-inflammatory effects, lidocaine has a potential to prevent AKI in dogs with GDV. Design and setting: Prospective, observational cohort study in client-owned dogs with GDV. Objective: To determine concentrations of renal biomarkers for AKI in dogs with GDV with and without intravenous (IV) lidocaine therapy. Methods: Thirty-two dogs were randomized to receive either IV lidocaine (2 mg/kg, followed by a lidocaine constant rate infusion at a dose of 50 µg/kg/min over 24 h; n = 17) or no lidocaine (n = 15). Blood and urine samples were taken at admission (T 0) (only blood), during or immediately after surgery (T 1), and 24 (T 24) and 48 (T 48) h after surgery. Plasma creatinine (pCr), plasma neutrophil gelatinase-associated lipocalin (pNGAL), urinary NGAL (uNGAL), uNGAL to creatinine ratio (UNCR), and urinary gamma-glutamyl transferase to creatinine ratio (uGGT/uCr) were evaluated. Biomarker concentrations were compared between dogs with and without IV lidocaine and the course of each marker was determined in comparison to its admission value. Results: In the entire population, a significantly higher pCr at T 0 (median, 95 µmol/L, interquartile range, 82-105) compared with T 1 (69 µmol/L, 60-78), T 24 (63 µmol/L, 52-78), and T 48 (78 µmol/L, 65-87) (P < 0.001) was found. Plasma NGAL increased significantly between T 0 (5.66 ng/mL, 3.58-7.43) and T 24 (7.50 ng/mL, 4.01-11.89) (P = 0.006) and T 48 (9.86 ng/mL, 5.52-13.92) (P < 0.001), respectively. Urinary NGAL increased significantly between T 1 (0.61 ng/mL, 0.30-2.59) and T 24 (2.62 ng/mL, 1.86-10.92) (P = 0.001) and T 48 (4.79 ng/mL, 1.96-34.97 (P < 0.001), respectively. UNCR increased significantly between T 1 (0.15 µg/mmol, 0.09-0.54) and T 24 (1.14 µg/mmol, 0.41-3.58) (P = 0.0015) and T 48 (1.34 µg/mmol, 0.30-7.42) (P < 0.001), respectively. Concentrations of uGGT/uCr increased significantly from T 0 highest at T 24 (6.20 U/mmol, 3.90-9.90) and significantly decreased at T 48 (3.76 U/mmol, 2.84-6.22) (P < 0.001). No significant differences in any renal biomarker concentration were found between dogs with and without IV lidocaine therapy. Conclusion and clinical relevance: Plasma NGAL, uNGAL and UNCR remained increased up to 48 h post-surgery. No evidence of lidocaine-associated renoprotection was found.

A Therapeutic Extracorporeal Device for Specific Removal of Pathologic Asymmetric Dimethylarginine from the Blood.

INTRODUCTION: Blood levels of uremic toxin, asymmetric dimethylarginine (ADMA), are strongly associated with mortality in sepsis, renal failure, and cardiovascular and renal disease patients. METHODS: An extracorporeal approach to reduce pathological ADMA was developed. The dimethylarginine dimethylaminohydrolase (DDAH) was immobilized on agarose beads to prepare a cartridge. The efficacy of cartridge for ADMA lowering in blood was investigated. RESULTS: The DDAH beads and cartridge reduced ADMA from solution or plasma. The magnitude of ADMA removal was dependent on the quantity of DDAH linked to the beads and the flow rate. When tested in association with plasmapheresis, the DDAH-cartridge was highly effective in ADMA removal from the blood and improved the arginine/ADMA ratio in a pig model. CONCLUSION: A new, safe, and effective extracorporeal approach to lower ADMA was developed which may have application in improving outcomes in patients with vascular complications and risk of mortality associated with high ADMA.

Ultrasonographic diagnosis of a hair foreign body in the urinary bladder of a dog.

A 13-year-old male neutered mixed-breed dog with a history of gallbladder mucocele and urolithiasis was evaluated by ultrasound. Two hyperechoic, linear foreign bodies with no distal acoustic shadowing were detected in the urinary bladder and urethra. Following the ultrasound examination, the patient underwent cystoscopy, and two single hairs were found and successfully retrieved. Considering that urinary bladder foreign bodies may be a source for urinary tract infection and can act as a nidus for urocystolith formation, removal is recommended. This is the first published report describing ultrasonographic diagnosis of a hair foreign body in the canine urinary bladder and urethra.

Aortic Sca-1+ Progenitor Cells Arise from the Somitic Mesoderm Lineage in Mice.

Sca-1+ progenitor cells in the adult mouse aorta are known to generate vascular smooth muscle cells (VSMCs), but their embryological origins and temporal abundance are not known. Using tamoxifen-inducible Myf5-CreER mice, we demonstrate that Sca-1+ adult aortic cells arise from the somitic mesoderm beginning at E8.5 and continue throughout somitogenesis. Myf5 lineage-derived Sca-1+ cells greatly expand in situ, starting at 4 weeks of age, and become a major source of aortic Sca-1+ cells by 6 weeks of age. Myf5-derived adult aortic cells are capable of forming multicellular sphere-like structures in vitro and express the pluripotency marker Sox2. Exposure to transforming growth factor-ß3 induces these spheres to differentiate into calponin-expressing VSMCs. Pulse-chase experiments using tamoxifen-inducible Sox2-CreERT2 mice at 8 weeks of age demonstrate that ∼35% of all adult aortic Sca-1+ cells are derived from Sox2+ cells. The present study demonstrates that aortic Sca-1+ progenitor cells are derived from the somitic mesoderm formed at the earliest stages of somitogenesis and from Sox2-expressing progenitors in adult mice.

Clinical and histopathologic findings in dogs with the ultrasonographic appearance of gastric muscularis unorganized hyperechoic striations.

BACKGROUND: Ultrasonographic appearance of unorganized hyperechoic striations (UHS) has been observed in the canine gastric muscularis layer. The purpose of the study was to determine the prevalence, sonographic and postmortem histologic features, and to determine the clinical significance of canine gastric muscularis UHS. In the prospective study, 72 dogs were included. The presence of gastric muscularis UHS were reviewed to determine its distribution and location. In the retrospective study, 167 dogs that had both abdominal ultrasonography and necropsy were included. RESULTS: The prevalence of gastric muscularis UHS in dogs was 37.5% in the prospective and 5.4% in the retrospective studies respectively. The higher prevalence in prospective study was due to greater anticipation by the radiologists in search for gastric muscularis UHS. In the ventral gastric wall, the muscularis UHS were better defined when the gastric lumen was empty or non-distended, and were mostly parallel with the serosa when the gastric wall was distended (with gas or fluid). Visualization of the dorsal gastric wall was often obscured by gas shadowing from luminal gas. Histopathology was performed on eight dogs with gastric muscularis UHS, three of which had fibrous tissue observed with Masson's trichrome stain. CONCLUSION: Presence of gastric muscularis UHS in dogs may have been attributable to presence of incomplete interfaces between the inner oblique, middle circular and outer longitudinal layers of the gastric tunica muscularis or due to presence of fibrous tissue within the gastric muscularis layer. The clinical significance of canine gastric muscularis UHS is uncertain.

Transcutaneous glomerular filtration rate measurement in a canine animal model of chronic kidney disease.

INTRODUCTION: Quantitative assessment of renal function by measurement of glomerular filtration rate (GFR) is an important part of safety and efficacy evaluation in preclinical drug development. Existing methods are often time consuming, imprecise and associated with animal burden. Here we describe the comparison between GFR determinations with sinistrin (PS-GFR) and fluorescence-labelled sinistrin-application and its transcutaneous detection (TD-GFR) in a large animal model of chronic kidney disease (CKD). METHODS: TD-GFR measurements compared to a standard method using i.v. sinistrin were performed in a canine model. Animals were treated with one-sided renal wrapping (RW) followed by renal artery occlusion (RO). Biomarker and remote hemodynamic measurements were performed. Plasma sinistrin in comparison to transcutaneous derived GFR data were determined during healthy conditions, after RW and RW+RO. RESULTS: RW alone did not led to any significant changes in renal function, neither with PS-GFR nor TD-GFR. Additional RO showed a rise in blood pressure (+68.0mmHg), plasma urea (+28.8mmol/l), creatinine (+224,4µmol/l) and symmetric dimethylarginine (SDMA™; +12.6µg/dl). Plasma sinistrin derived data confirmed the expected drop (-44.7%, p<0.0001) in GFR. The calculated transcutaneous determined Fluorescein Isothiocyanate (FITC)-sinistrin GFR showed no differences to plasma sinistrin GFR at all times. Both methods were equaly sensitive to diagnose renal dysfunction in the affected animals. DISCUSSION: Renal function assessment using TD-GFR is a valid method to improve preclinical drug discovery and development. Furthermore, TD-GFR method offers advantages in terms of reduced need for blood sampling and thus decreasing animal burden compared to standard procedures.

Serum anti-Müllerian hormone concentrations before and after treatment of an ovarian granulosa cell tumour in a cat.

CASE SUMMARY: A 15-year-old female cat was presented for investigation of progressive behavioural changes, polyuria, polydipsia and periuria. An ovarian granulosa cell tumour was identified and the cat underwent therapeutic ovariohysterectomy (OHE). The cat's clinical signs resolved, but 6 months later it was diagnosed as having an anaplastic astrocytoma and was euthanased. Serum anti-Müllerian hormone (AMH) concentration prior to OHE was increased vs a control group of entire and neutered female cats. Following OHE, serum AMH concentration decreased to <1% of the original value. RELEVANCE AND NOVEL INFORMATION: Serum AMH measurement may represent a novel diagnostic and monitoring tool for functional ovarian neoplasms in cats.

Skin-derived precursors from human subjects with Type 2 diabetes yield dysfunctional vascular smooth muscle cells.

Objective: Few methods enable molecular and cellular studies of vascular aging or Type 2 diabetes (T2D). Here, we report a new approach to studying human vascular smooth muscle cell (VSMC) pathophysiology by examining VSMCs differentiated from progenitors found in skin. Approach and results: Skin-derived precursors (SKPs) were cultured from biopsies (N=164, ∼1 cm2) taken from the edges of surgical incisions of older adults (N=158; males 72%; mean age 62.7 ± 13 years) undergoing cardiothoracic surgery, and differentiated into VSMCs at high efficiency (>80% yield). The number of SKPs isolated from subjects with T2D was ∼50% lower than those without T2D (cells/g: 0.18 ± 0.03, N=58 versus 0.40 ± 0.05, N=100, P<0.05). Importantly, SKP-derived VSMCs from subjects with T2D had higher Fluo-5F-determined baseline cytosolic Ca2+ concentrations (AU: 1,968 ± 160, N=7 versus 1,386 ± 170, N=13, P<0.05), and a trend toward greater Ca2+ cycling responses to norepinephrine (NE) (AUC: 177,207 ± 24,669, N=7 versus 101,537 ± 15,881, N=20, P<0.08) despite a reduced frequency of Ca2+ cycling (events s-1 cell-1: 0.011 ± 0.004, N=8 versus 0.021 ± 0.003, N=19, P<0.05) than those without T2D. SKP-derived VSMCs from subjects with T2D also manifest enhanced sensitivity to phenylephrine (PE) in an impedance-based assay (EC50 nM: 72.3 ± 63.6, N=5 versus 3,684 ± 3,122, N=9, P<0.05), and impaired wound closure in vitro (% closure: 21.9 ± 3.6, N=4 versus 67.0 ± 10.3, N=4, P<0.05). Compared with aortic- and saphenous vein-derived primary VSMCs, SKP-derived VSMCs are functionally distinct, but mirror defects of T2D also exhibited by primary VSMCs. CONCLUSION: Skin biopsies from older adults yield sufficient SKPs to differentiate VSMCs, which reveal abnormal phenotypes of T2D that survive differentiation and persist even after long-term normoglycemic culture.

Enhanced Cardiac Differentiation of Human Cardiovascular Disease Patient-Specific Induced Pluripotent Stem Cells by Applying Unidirectional Electrical Pulses Using Aligned Electroactive Nanofibrous Scaffolds.

In the embryonic heart, electrical impulses propagate in a unidirectional manner from the sinus venosus and appear to be involved in cardiogenesis. In this work, aligned and random polyaniline/polyetersulfone (PANI/PES) nanofibrous scaffolds doped by Camphor-10-sulfonic acid (ß) (CPSA) were fabricated via electrospinning and used to conduct electrical impulses in a unidirectional and multidirectional fashion, respectively. A bioreactor was subsequently engineered to apply electrical impulses to cells cultured on PANI/PES scaffolds. We established cardiovascular disease-specific induced pluripotent stem cells (CVD-iPSCs) from the fibroblasts of patients undergoing cardiothoracic surgeries. The CVD-iPSCs were seeded onto the scaffolds, cultured in cardiomyocyte-inducing factors, and exposed to electrical impulses for 1 h/day, over a 15-day time period in the bioreactor. The application of the unidirectional electrical stimulation to the cells significantly increased the number of cardiac Troponin T (cTnT+) cells in comparison to multidirectional electrical stimulation using random fibrous scaffolds. This was confirmed by real-time polymerase chain reaction for cardiac-related transcription factors (NKX2.5, GATA4, and NPPA) and a cardiac-specific structural gene (TNNT2). Here we report for the first time that applying electrical pulses in a unidirectional manner mimicking the unidirectional wave of electrical stimulation in the heart, could increase the derivation of cardiomyocytes from CVD-iPSCs.

Vascular smooth muscle cell differentiation from human stem/progenitor cells.

Transplantation of vascular smooth muscle cells (VSMCs) is a promising cellular therapy to promote angiogenesis and wound healing. However, VSMCs are derived from diverse embryonic sources which may influence their role in the development of vascular disease and in its therapeutic modulation. Despite progress in understanding the mechanisms of VSMC differentiation, there remains a shortage of robust methods for generating lineage-specific VSMCs from pluripotent and adult stem/progenitor cells in serum-free conditions. Here we describe a method for differentiating pluripotent stem cells, such as embryonic and induced pluripotent stem cells, as well as skin-derived precursors, into lateral plate-derived VSMCs including 'coronary-like' VSMCs and neural crest-derived VSMC, respectively. We believe this approach will have broad applications in modeling origin-specific disease vulnerability and in developing personalized cell-based vascular grafts for regenerative medicine.

Use of a Microsoft Excel based add-in program to calculate plasma sinistrin clearance by a two-compartment model analysis in dogs.

Assessment of renal function by means of plasma clearance of a suitable marker has become standard procedure for estimation of glomerular filtration rate (GFR). Sinistrin, a polyfructan solely cleared by the kidney, is often used for this purpose. Pharmacokinetic modeling using adequate software is necessary to calculate disappearance rate and half-life of sinistrin. The purpose of this study was to describe the use of a Microsoft excel based add-in program to calculate plasma sinistrin clearance, as well as additional pharmacokinetic parameters such as transfer rates (k), half-life (t1/2) and volume of distribution (Vss) for sinistrin in dogs with varying degrees of renal function.

Extrahepatic biliary atresia in a 4-week-old Pug.

OBJECTIVE: To report diagnosis and surgical therapy of extrahepatic biliary atresia in a dog. STUDY DESIGN: Clinical report. ANIMAL: Dog (n = 1). METHODS: A 4-week-old female intact Pug presented with acholic feces, lethargy, and poor weight gain. On the basis of the stool color and abdominal ultrasonography findings, extrahepatic biliary atresia was suspected. Intraoperatively, no major duodenal papilla was identified and cholecystoduodenostomy was performed. RESULTS: One day postoperatively the stool color was considered normal. On ultrasonographic examinations up to 6 months after surgery, the gall bladder remained small and the stoma was functional. Serum biochemical profile after 10 months had a mild increase in alanine-aminotransferase (149 U/L; reference interval [RI], 0-85 U/L) whereas alkaline phosphatase and glutamate dehydrogenase were within normal limits. At 15 months, clinical examination was normal and blood ammonia concentration was within normal limits (43 µmol/L; RI: <100 µmol/L) but bile acid concentrations were still markedly elevated (fasting, 95 µmol/L [RI: <20 µmol/L] to postprandial, 127 µmol/L [RI: <35 µmol/L]). CONCLUSIONS: Ultrasonography and stool color can be helpful noninvasive diagnostic evidence in extrahepatic biliary atresia. Surgical correction with cholecystoduodenostomy with a good clinical outcome is possible even in very young dogs. Long-term prognosis in dogs with extrahepatic biliary atresia is unknown.

A pilot study to assess the feasibility of transcutaneous glomerular filtration rate measurement using fluorescence-labelled sinistrin in dogs and cats.

In dogs and cats an assessment of renal function is often needed, however, existing methods including urine and plasma clearances are invasive, cumbersome and time consuming. This pilot study evaluated the feasibility of a transcutaneous glomerular filtration rate (GFR) measurement in dogs and cats. Additionally the optimal dose and location for the transcutaneous measurement device were investigated. Renal elimination of fluorescein-isothiocyanate-labelled sinistrin (FITC-S) was measured transcutaneously for 4 hours. The procedures were performed in awake, freely moving animals using escalating doses of FITC-S (10 mg/kg, 30 mg/kg, 50 mg/kg) with a wash-out period of at least 24 h in between. Multiple devices were placed on each animal. The resulting FITC-S disappearance curves were visually assessed to determine the most suitable location and the appropriate dose to reach an adequate transcutaneous peak signal for kinetic analysis. In both species 30 mg/kg were adequate for kinetic calculation. The most suitable place for the device was the lateral thoracic wall in dogs and the ventral abdominal wall in cats, respectively. Transcutaneous FITC-S clearance was then repeated using the optimal dose and location and in parallel with an additional plasma sinistrin clearance. Plasma elimination half-lives [min] were 26, 31 and 35, and corresponding transcutaneous elimination half-lives [min] were 26, 34 and 55, respectively in the dogs. Plasma elimination half-lives [min] were 51, 60 and 61, and corresponding transcutaneous elimination half-lives [min] were 75, 96 and 83, respectively in the cats. In conclusion, transcutaneous FITC-S clearance is a feasible method for the assessment of GFR in awake dogs and cats. It is noninvasive, well tolerated and easy to perform even in a clinical setting with results being readily available. A dose of 30 mg/kg of FITC-S seems adequate for kinetic assessment. Further studies are now needed to establish reference values and evaluate transcutaneous renal clearance in various conditions.

Serum-free differentiation of functional human coronary-like vascular smooth muscle cells from embryonic stem cells.

AIMS: Despite the diverse developmental origins of vascular smooth muscle cells (VSMCs), recent attempts to generate VSMCs from human embryonic stem cells (hESCs) differentiated along various lineages did not yield distinct cell phenotypes. The aim of this study was to derive and characterize functional coronary-like VSMCs from hESCs using serum-free cardiac-directed differentiation. METHODS AND RESULTS: Embryoid bodies (EBs) from three pluripotent stem cell lines subjected to cardiac-directed differentiation in defined media were characterized over 30 days for VSMC-specific gene expression by qRT-PCR, immunofluorescence microscopy and fluorescence-activated cell sorting (FACS). EBs composed of cardiomyocytes, endothelial cells (ECs), fibroblasts, and VSMCs underwent FACS on d28 to reveal that the VSMCs form a distinct subpopulation, which migrate with ECs in an in vitro angiogenesis assay. To enrich for VSMCs, d28 EBs were dissociated and cultured as monolayers. Over several passages, mRNA and protein levels of cardiomyocyte, endothelial, and fibroblast markers were abolished, whereas those of mature VSMCs were unchanged. Vascular endothelial growth factor and basic fibroblast growth factor were critical for the separation of the cardiac and VSMC lineages in EBs, and for the enrichment of functional VSMCs in monolayer cultures. Calcium cycling and cell shortening responses to vasoconstrictors in hESC-derived VSMCs in vitro were indistinguishable from primary human coronary artery SMCs, and distinct from bladder and aorta SMCs. VSMCs identically derived from green fluorescent protein -expressing hESCs integrated in and contributed to new vessel formation in vivo. CONCLUSION: The ability to generate hESC-derived functional human coronary-like VSMCs in serum-free conditions has implications for disease modelling, drug screening, and regenerative therapies.

Directed differentiation of skin-derived precursors into functional vascular smooth muscle cells.

OBJECTIVE: The goal of this study was to characterize the factors and conditions required for smooth muscle cell (SMC)-directed differentiation of Sox2(+) multipotent rat and human skin-derived precursors (SKPs) and to define whether they represent a source of fully functional vascular SMCs for applications in vivo. METHODS AND RESULTS: We found that rat SKPs can differentiate almost exclusively into SMCs by reducing serum concentrations to 0.5% to 2% and plating them at low density. Human SKPs derived from foreskin required the addition of transforming growth factor-ß1 or -ß3 to differentiate into SMCs, but they did so even in the absence of serum. SMC formation was confirmed by quantitative reverse transcription-polymerase chain reaction, immunocytochemistry, and fluorescence-activated cell sorting, with increased expression of smoothelin-B and little to no expression of telokin or smooth muscle γ-actin, together indicating that SKPs differentiated into vascular rather than visceral SMCs. Rat and human SKP-derived SMCs were able to contract in vitro and also wrap around and support new capillary and larger blood vessel formation in angiogenesis assays in vivo. CONCLUSIONS: SKPs are Sox2(+) progenitors that represent an attainable autologous source of stem cells that can be easily differentiated into functional vascular SMCs in defined serum-free conditions without reprogramming. SKPs represent a clinically viable cell source for potential therapeutic applications in neovascularization.

Peptide-mediated disruption of calmodulin-cyclin E interactions inhibits proliferation of vascular smooth muscle cells and neointima formation.

RATIONALE: Cell cycle progression in vascular smooth muscle cells (VSMCs) is a therapeutic target for restenosis. OBJECTIVE: Having discovered that calmodulin (CaM)-dependent cyclin E/CDK2 activity underlies Ca(2+)-sensitive G(1)-to-S phase transitions in VSMCs, we sought to explore the physiological importance of the CaM-cyclin E interaction. METHODS AND RESULTS: A peptide based on the CaM binding sequence (CBS) of cyclin E was designed to interfere with CaM-cyclin E binding. Compared with control peptides, CBS blocked activating Thr160 phosphorylation of CDK2, decreased basal cyclin E/CDK2 activity, and eliminated Ca(2+)-sensitive cyclin E/CDK2 activity in nuclear extracts from mouse VSMCs. Nucleofection with CBS, or treatment with CBS conjugated to the HIV-1 TAT protein transduction domain to improve bioavailability, inhibited G(1)-to-S cell cycle progression in a dose-dependent manner. These effects were not observed with control peptides. TAT-CBS inhibited (3)H-thymidine incorporation in primary human aortic SMCs (HA-SMCs) in vitro, manifested greater transduction into HA-SMCs compared with endothelial cells in vitro, and limited decreased SM22α expression, neointima formation, and medial thickening without affecting collagen deposition or reendothelialization in a mouse model of carotid artery injury in vivo. The antiproliferative effects of CBS remained evident in mouse embryonic fibroblasts derived from wild-type mice but not cyclin E1/E2 double knockout mice. CONCLUSIONS: A synthetic peptide designed to disrupt CaM-cyclin E binding inhibits Ca(2+)/CaM-dependent CDK2 activity, cell cycle progression, and proliferation in VSMCs and limits arterial remodeling following injury. Importantly, this effect appears to be cyclin E-dependent and may form the basis of a potentially novel therapeutic approach for restenosis.