Fatty acid analysis in serum of patients with elevated lipoprotein(a) and cardiovascular disease undergoing lipoprotein apheresis.

BACKGROUND: Lipoprotein apheresis (LA) is an extracorporeal treatment that transiently reduces lipoprotein (a) by 60% and leads to an 80-92% reduction in major adverse cardiovascular events. LA has a significant impact on lipid profile in serum of patients with atherosclerotic cardiovascular disease. OBJECTIVE: To investigate the effects of LA on the composition of serum fatty acids (FAs), focusing on those which could have an impact on cardiovascular disease (CVD). METHODS: This is a prospective study in the First Department of Cardiology of the Medical University of Gdansk, Poland. Serum samples were collected from 28 patients before LA, just after the procedure, and 7 days after LA. Additionally, in a smaller group of patients, the samples were collected after second tour of LA (2 weeks later), as well as after 1 year from the first procedure. The serum FA profile was analyzed using gas chromatography-mass spectrometry. RESULTS: After the LA procedure, a substantial change in serum FA composition along with LDL-C and Lp(a) decrease were observed 7 days after procedure, but these parameters returned to the values similar to those before procedure after 14 days. Very long-chain FAs (VLCFAs) and very long-chain monounsaturated FAs (VLC-MUFAs) were eluted at 57% and remained low even 7 days after LA (p=0.027 and p < 0.001, respectively). We also observed an increase in the percentage of total branched-chain FAs (BCFAs) (p=0.004) and anteiso BCFAs (p=0.012) after FA. After 1 year of regular LA, a substantial decrease in serum VLC-MUFAs and n3 polyunsaturated FA (PUFAs) were noted. CONCLUSIONS: Decreased VLCFAs and VLC-MUFAs involved in CVD development remained low even 7 days after LA. An acute increase in the levels of anti-inflammatory BCFAs was observed. In turn long-term regular administration of LA substantially decreased VLC-MUFA and n3 PUFA.

Isolation, expansion and characterization of porcine urinary bladder smooth muscle cells for tissue engineering.

BACKGROUND: A key requirements for therapy utilizing the tissue engineering methodologies is use of techniques which have the capability to yield a high number of cells, from small tissue biopsy in a relatively short time. Up to date there was no optimal methods of isolation and expansion of urinary bladder smooth muscle cells (UB-SMCs). The aim of this study was to compare isolation and expansion techniques of UB-SMCs to select the most repeatable and efficient one. METHOD: Five protocols of porcine UB- SMCs isolation including enzymatic and explant techniques and three expansion techniques were compared. Isolation effectiveness was evaluated using trypan blue assay. Cell phenotype was confirmed by immunofluorescence staining. Proliferation rate was analyzed using MTT and X- Celligence system. Cellular senescence was assessed measuring ß-galactosidase activity. RESULTS: Enzymatic methods using collagenase with dispase (method I) or collagenase only (method III) allowed to isolate much larger number of cells than the methods using trypsin with collagenase (method II) and collagenase after digestion with trypsin (method IV). The success rate of establishment of primary culture was the highest when the isolated cells were cultured in the Smooth muscle Growth Medium-2 (SmGM-2). Expression of the smooth muscle markers- alpha smooth muscle actin and smoothelin was the highest for cells isolated by enzymatic method I and cultured in SmGM-2. There was no significant signs of cell senescence until the 8th passage. CONCLUSION: The most efficient method of establishment of porcine UB-SMCs culture is enzymatic digestion of urinary bladder tissue with collagenase and dispase and culture of isolated cells in SmGM-2. This method was up to 10 times more efficient than other methods used for isolation and culture of UB-SMCs. This is an easy and consistent method for obtaining high numbers of urinary bladder smooth muscle cells.

Optimization of porcine urothelial cell cultures: Best practices, recommendations, and threats.

Many experimental approaches have been conducted in order to isolate urothelial cells from bladder tissue biopsies, but each method described has utilized different protocols and sources of bladder tissue. In this study, we compared the different methods of urothelial cell isolation available in literature together with standardized methods in order to obtain more unified results. Five methods for primary porcine urothelial culture establishment were compared: tissue explants and four enzymatic methods utilizing collagenase II, dispase II, combination of dispase II and trypsin, and trypsin alone. The average number of isolated cells, cell morphology, success of established culture, average number of cells from the first passage, expression of p63 and pancytokeratin and the characterization of urothelial cell growth, and aging were analyzed during the in vitro culture. The method utilizing dispase II was the most efficient and reproducible method for the isolation and culture of porcine urothelial cells when compared to the other tested methods. Urothelial cells obtained by this method grew considerably well and the cultures were established with high efficiency, which enabled us in obtaining a large quantity of cells with normal morphology. Contamination with fibroblasts in this method was the lowest. The utilization of a proper method for urothelial cell isolation is a critical step in the urinary tract regeneration when using tissue engineering techniques. In summary, this study demonstrated that by utilizing the described method with dispase II, a suitable number of cells was achieved, proving the method useful for tissue regeneration.

Targeted therapy for stress urinary incontinence: a systematic review based on clinical trials.

INTRODUCTION: Controversy exists regarding the therapeutic benefit of cell-based therapy in the treatment of stress urinary incontinence (SUI). AREAS COVERED: The aim of this systematic review was to evaluate evidence regarding the therapeutic effect and safety of cell-based therapy in the treatment of SUI and to propose a new approach to SUI treatment utilizing tissue engineering methodologies. We have thoroughly reviewed the literature using PubMed in order to identify only original, clinical studies involving cell therapy for SUI. EXPERT OPINION: Cell-based therapy, as practiced today, is a safe but ineffective method for SUI treatment. The key to an optimal therapeutic outcome in SUI is accurate diagnosis combined with targeted therapy. Targeted therapy in SUI should be based on cell implantation to restore and regenerate the damaged urethral sphincter and/or the construction of a neo-pubourethral ligament utilizing tissue engineering methodologies.