Andrzej Rutkowski

Heme Oxygenase-1 and the Vascular Bed: From Molecular Mechanisms to Therapeutic Opportunities

Heme oxygenase-1, an enzyme degrading heme to carbon monoxide, iron, and biliverdin, has been recognized as playing a crucial role in cellular defense against stressful conditions, not only related to heme release. HO-1 protects endothelial cells from apoptosis, is involved in blood-vessel relaxation regulating vascular tone, attenuates inflammatory response in the vessel wall, and participates in blood-vessel formation by means of angiogenesis and vasculogenesis. The latter functions link HO-1 not only to cardiovascular ischemia but also to many other conditions that, like development, wound healing, or cancer, are dependent on neovascularization. The aim of this comprehensive review is to address the mechanisms of HO-1 regulation and function in cardiovascular physiology and pathology and to demonstrate some possible applications of the vast knowledge generated so far. Recent data provide powerful evidence for the involvement of HO-1 in the therapeutic effect of drugs used in cardiovascular diseases. Novel studies open the possibilities of application of HO-1 for gene and cell therapy. Therefore, research in forthcoming years should help to elucidate both the real role of HO-1 in the effect of drugs and the clinical feasibility of HO-1-based cell and gene therapy, creating the effective therapeutic avenues for this refined antioxidant system.

Heme Oxygenase-1 Accelerates Cutaneous Wound Healing in Mice

Heme oxygenase-1 (HO-1), a cytoprotective, pro-angiogenic and anti-inflammatory enzyme, is strongly induced in injured tissues. Our aim was to clarify its role in cutaneous wound healing. In wild type mice, maximal expression of HO-1 in the skin was observed on the 2nd and 3rd days after wounding. Inhibition of HO-1 by tin protoporphyrin-IX resulted in retardation of wound closure. Healing was also delayed in HO-1 deficient mice, where lack of HO-1 could lead to complete suppression of reepithelialization and to formation of extensive skin lesions, accompanied by impaired neovascularization. Experiments performed in transgenic mice bearing HO-1 under control of keratin 14 promoter showed that increased level of HO-1 in keratinocytes is enough to improve the neovascularization and hasten the closure of wounds. Importantly, induction of HO-1 in wounded skin was relatively weak and delayed in diabetic (db/db) mice, in which also angiogenesis and wound closure were impaired. In such animals local delivery of HO-1 transgene using adenoviral vectors accelerated the wound healing and increased the vascularization. In summary, induction of HO-1 is necessary for efficient wound closure and neovascularization. Impaired wound healing in diabetic mice may be associated with delayed HO-1 upregulation and can be improved by HO-1 gene transfer.

Pooled sample-based GWAS: a cost-effective alternative for identifying colorectal and prostate cancer risk variants in the Polish population.

Background Prostate cancer (PCa) and colorectal cancer (CRC) are the most commonly diagnosed cancers and cancer-related causes of death in Poland. To date, numerous single nucleotide polymorphisms (SNPs) associated with susceptibility to both cancer types have been identified, but their effect on disease risk may differ among populations. Methods To identify new SNPs associated with PCa and CRC in the Polish population, a genome-wide association study (GWAS) was performed using DNA sample pools on Affymetrix Genome-Wide Human SNP 6.0 arrays. A total of 135 PCa patients and 270 healthy men (PCa sub-study) and 525 patients with adenoma (AD), 630 patients with CRC and 690 controls (AD/CRC sub-study) were included in the analysis. Allele frequency distributions were compared with t-tests and χ2-tests. Only those significantly associated SNPs with a proxy SNP (p<0.001; distance of 100 kb; r2>0.7) were selected. GWAS marker selection was conducted using PLINK. The study was replicated using extended cohorts of patients and controls. The association with previously reported PCa and CRC susceptibility variants was also examined. Individual patients were genotyped using TaqMan SNP Genotyping Assays. Results The GWAS selected six and 24 new candidate SNPs associated with PCa and CRC susceptibility, respectively. In the replication study, 17 of these associations were confirmed as significant in additive model of inheritance. Seven of them remained significant after correction for multiple hypothesis testing. Additionally, 17 previously reported risk variants have been identified, five of which remained significant after correction. Conclusion Pooled-DNA GWAS enabled the identification of new susceptibility loci for CRC in the Polish population. Previously reported CRC and PCa predisposition variants were also identified, validating the global nature of their associations. Further independent replication studies are required to confirm significance of the newly uncovered candidate susceptibility loci.

CD45-CD14 +CD34 + murine bone marrow low-adherent mesenchymal primitive cells preserve multilineage differentiation potential in long-term in vitro culture.

Bone marrow-derived cells have been postulated as a source of multipotent mesenchymal stem cells (MSC). However, the whole fraction of MSC remains heterogeneous and the expansion of primitive subset of these cells is still not well established. Here, we optimized the protocol for propagating the low-adherent subfraction of MSC which results in long-term expansion of population characterized by CD45−CD14+CD34+ phenotype along with expression of common MSC markers. We established that the expanded MSC are capable of differentiating into endothelial cells highly expressing angiogenic markers and exhibiting functional properties of endothelium. Moreover, we found these cells to be multipotent and capable of giving rise into cells from neuronal lineages. Interestingly, the expanded MSC form characteristic cellular spheres in vitro indicating primitive features of these cells. In sum, we isolated the novel multipotent subpopulation of CD45−CD14+ CD34+ bone marrow-derived cells that could be maintained in long-term culture without losing this potential.

Preoperative radiotherapy and local excision of rectal cancer: Long-term results of a randomised study

BACKGROUND AND PURPOSE It is uncertain whether local control is acceptable after preoperative radiotherapy and local excision (LE). An optimal preoperative dose/fractionation schedule has not yet been established. MATERIAL AND METHODS In a phase III study, patients with cT1-2N0M0 or borderline cT2/T3N0M0 < 4 cm rectal adenocarcinomas were randomised to receive either 5 × 5 Gy plus 1 × 4 Gy boost or chemoradiation: 50.4 Gy in 28 fractions plus 3 × 1.8 Gy boost and 5-fluorouracil with leucovorin bolus. LE was performed 6-8 weeks later. Patients with ypT0-1R0 disease were observed. Completion total mesorectal excision (CTME) was recommended for poor responders, i.e. ypT1R1/ypT2-3. RESULTS Of 61 randomised patients, 10 were excluded leaving 51 for analysis; 29 in the short-course group and 22 in the chemoradiation group. YpT0-1R0 was observed in 66% of patients in the short-course group and in 86% in the chemoradiation group, p = 0.11. CTME was performed only in 46% of patients with ypT1R1/ypT2-3. The median follow-up was 8.7 years. Local recurrence incidences and overall survival at 10 years were respectively for the short-course group vs. the chemoradiation group 35% vs. 5%, p = 0.036 and 47% vs. 86%, p = 0.009. In total, local recurrence at 10 years was 79% for ypT1R1/T2-3 without CTME. CONCLUSIONS This trial suggests that in the LE setting, both local recurrence and survival are worse after short-course radiotherapy than after chemoradiation. Because of the risk of bias, a confirmatory study is desirable. Lack of CTME is associated with an unacceptably high local recurrence rate.

Exome scale map of genetic alterations promoting metastasis in colorectal cancer

BackgroundApproximately 90% of colorectal cancer (CRC) deaths are caused by tumors ability to migrate into the adjacent tissues and metastase into distant organs. More than 40 genes have been causally linked to the development of CRC but no mutations have been associated with metastasis yet. To identify molecular basis of CRC metastasis we performed whole-exome and genome-scale transcriptome sequencing of 7 liver metastases along with their matched primary tumours and normal tissue. Multiple, spatially separated fragments of primary tumours were analyzed in each case. Uniformly malignant tissue specimen were selected with macrodissection, for three samples followed with laser microdissection.Results> 100 sequencing coverage allowed for detection of genetic alterations in subpopulation of tumour cells. Mutations in KRAS, APC, POLE, and PTPRT, previously associated with CRC development, were detected in most patients. Several new associations were identified, including PLXND1, CELSR3, BAHD1 and PNPLA6.ConclusionsWe confirm the essential role of inflammation in CRC progression but question the mechanism of matrix metalloproteinases activation described in other work. Comprehensive sequencing data made it possible to associate genome-scale mutation distribution with gene expression patterns. To our knowledge, this is the first work to report such link in CRC metastasis context.

The safety of low-molecular-weight heparins in the prevention of venous thromboembolism in surgically-treated cancer patients: results of a multicentre observational study

Aim of the study Despite widespread use of pharmacological prophylaxis, venous thromboembolism (VTE) still constitutes a common complication in cancer patients. The aim of the study was to analyse the safety of low-molecular-weight heparins (LMWH) in the prevention of VTE in surgically-treated cancer patients. Material and methods A total of 5207 cancer patients (44.5% men and 55.5% women) aged 16–97 years participated in a prospective observational study conducted in 13 Polish cancer centres in 2005–2008. This cohort included 4782 subjects who were treated surgically and received LMWH as a pharmacological prophylaxis for VTE prior to or after the surgery. The incidence of haemorrhagic complications and thrombocytopaenia was analysed in this cohort, along with intra-hospital mortality. Results Mean duration of LMWH administration was 9.4 ±7.8 days. Haemorrhagic complications: heavy (n = 15) or light bleeding (n = 299), were observed in 314 patients (6.5%). A total of 314 patients (6.5%) presented with haemorrhagic complications: heavy (n = 15, 0.3%) or light bleeding (n = 299, 6.3%). Four cases of heavy bleeding: gastrointestinal bleeding (n = 2), retroperitoneal bleeding (n = 1), and central nervous system bleeding (n = 1), were classified as definitely related to LMWH. No significant association was found between the incidence of haemorrhagic complications and the type of administered LWMH (p = 0.523). No cases of thrombocytopaenia or deaths related to administration of LMWH were reported. Conclusions LMWH seems to be a safe form of pharmacological prophylaxis for VTE in surgically-treated cancer patients.

Prospective monitoring of the early treatment results of the GI tract tumours at the Oncology Centre in Warsaw

The current treatment of GI tract tumours is based to a large degree on the combined use of varied types of therapy: surgery, chemotherapy, irradiation. Severe post-operative complications may significantly affect the possibilities of application of non-surgical methods of treatment. Therefore, monitoring the early results of surgical treatment is extremely crucial to ensuring the appropriate necessary follow up and keep the risk of post-operative complications to a minimum. This paper describes the methodology of prospective collection of clinical data, which allows monitoring the quality of the surgical treatment of GI tumours on a current basis. On the basis of the data collected in the period from 2010 to 2015, the results of the analysis of three types of surgeries were presented (anterior resection of the anus, right hemicolectomy and a total gastric resection) and the actions taken on the basis of the annual report concerning the post-operative complication risks were described. Moreover, the examples of the application of prospectively collected clinical information for academic purposes were presented.