Evaluation of cardiac muscle microvessel density in children diagnosed with cyanotic heart defects

Abstract: Angiogenesis is largely an adaptive response to tissue hypoxia, which occurs in a wide variety of situations. Interestingly, the extent of hypoxia-induces angiogenesis in the cardiac muscle of children diagnosed with congenital cyanotic heart defects is not well established. Thus, the aim of this study was to 1) estimate the cardiac muscle microvessel density (MVD) in children diagnosed with cyanotic (study group) and non-cyanotic (control group) heart defects and to 2) evaluate the prognostic significance of MVD value in the development of ventricular dysfunction in the postoperative period. The study group included 42 children diagnosed with cyanotic heart defects. The control group comprised 33 patients with a diagnosis of non-cyanotic heart failure. The collected tissue included cardiac muscle sections from the right atrium and interventricular or interatrial wall during surgical correction of the defect. Immunocytochemistry with monoclonal mouse anti-human antibodies against CD31, CD34 and CD105 was employed to estimate the MVD value. The mean cardiac muscle MVD, defined by CD34 expression, was 596.7 ± 32.6 microvessels per 1 mm2 in the study group, which was notsignificantly different from the mean MVD in the control group (461.2 ± 30.5). Interestingly, in non-cyanotic heart defects, an inner area of subendocardial meshwork was estimated to have 75.3 ± 7.0 microvessels per 1 mm2, compared to 92.8 ± 10.9 microvessels per 1 mm2 (p = 0.0082) in patients with cyanotic heart defects. No significant correlations between MVD value and ventricular dysfunction were found. Cyanotic heart defects resulting in chronic hypoxia might provoke angiogenesis in the subendocardial meshwork of the heart wall. The process seems to be independent of the type of cyanotic heart disease and most likely takes place during antenatal development. A ventricular dysfunction observed in some cases of cyanotic heart defects could not be predicted by the estimation of MVD

Disturbances in angiogenesis and vascular maturation in the skin are associated with diabetic kidney disease in type 1 diabetes

Introduction. The skin, as one of the most accessible tissues, is frequently used for investigations of microcirculation and angiogenesis. The aim of this study was to assess the relationship between the dermal microvessel density (MVD) and maturity and the presence of diabetic kidney disease (DKD) in adults with type 1 diabetes (T1D). Skin as the most accessible organ served as a model for the study of angiogenesis. Materials and methods. 148 consecutive T1D patients (87 men), median age of 41 [interquartile range (IQR): 31–49] years and diabetes duration of 21 (17–30) years, participated in the study. The patients were under the care of the Department of Internal Medicine and Diabetology, Poznan University of Medical Sciences. Diabetic kidney disease was diagnosed in patients with increased albuminuria and at least 10-year duration of diabetes or evidence of diabetic retinopathy. The skin biopsy was performed on distal part of lower leg, using a sterile, disposable 3 mm biopsy punch with plunger (Disposable Biopsy Punches, Integra™ Miltex®). In the immunohistochemical analyses, we used: anti-CD133, anti-CD34, anti-CD31, and anti-von Willebrand factor (vWF) autoantibodies. Microvessel density measurement in all specimens was performed using “hot spots technique”. Slides were scanned using the MiraxMidi scanner (Carl Zeiss) and were viewed using CaseViewer (3DHISTECH Ltd. Budapest, Hungary). Data were analyzed using Statistica v. 13 software. Results. In the study group 21 patients with diagnosis DKD+, as compared to 127 subjects withaout DKD–, had longer duration of diabetes [30 (IQR: 21–36) vs. 21 (16–28) years, p = 0.002], higher prevalence of hypertension [14 (67%) vs. 37 (29%), p = 0.002], lower estimated glomerular filtration rate (eGFR) [66 (55–88) vs. 94 (83–106) mL/min/1.73 m2, p &lt; 0.001]. Median MVD compared between groups with and without DKD, was similar for CD34+ vessels/1 mm2 [123 (100–170) vs. 121 (100–170), p = 0.775], CD133+ vessels/1 mm2 [79 (50–100) vs. 79 (63–93), p = 0.823], and for CD31+ vessels/ 1 mm2 [29 (21–46) vs. 38 (17–58), p = 0.454]. Median MVD vWF+ vessels/1 mm2 was lower in the group with than without DKD: 42 (25–54) vs. 54 (43–71), p = 0.009. The values given above were calculated for both layers of the dermis (papillary and reticular dermis). In multivariate logistic regression analysis presence of diabetic kidney disease was associated with lower median vWF+ MVD [odds ratio: 0.97 (95% confidence interval: 0.95–0.99), p = 0.017], with adjustment for age, gender, eGFR value, diabetes duration and presence of hypertension. MVD did not differ significantly between chronic kidney disease stages. Conclusion. In patients with type 1 diabetes and diabetic kidney disease the disturbances in the angiogenesis and vascular maturation are present. The number of mature blood vessels (vWF+) in the skin is reduced. Disturbances in the angiogenesis occur at early stages of diabetic kidney disease.Introduction. The skin, as one of the most accessible tissues, is frequently used for investigations of microcirculation and angiogenesis. The aim of this study was to assess the relationship between the dermal microvessel density (MVD) and maturity and the presence of diabetic kidney disease (DKD) in adults with type 1 diabetes (T1D). Skin as the most accessible organ served as a model for the study of angiogenesis. Materials and methods. 148 consecutive T1D patients (87 men), median age of 41 [interquartile range (IQR): 31–49] years and diabetes duration of 21 (17–30) years, participated in the study. The patients were under the care of the Department of Internal Medicine and Diabetology, Poznan University of Medical Sciences. Diabetic kidney disease was diagnosed in patients with increased albuminuria and at least 10-year duration of diabetes or evidence of diabetic retinopathy. The skin biopsy was performed on distal part of lower leg, using a sterile, disposable 3 mm biopsy punch with plunger (Disposable Biopsy Punches, Integra™ Miltex®). In the immunohistochemical analyses, we used: anti-CD133, anti-CD34, anti-CD31, and anti-von Willebrand factor (vWF) autoantibodies. Microvessel density measurement in all specimens was performed using “hot spots technique”. Slides were scanned using the MiraxMidi scanner (Carl Zeiss) and were viewed using CaseViewer (3DHISTECH Ltd. Budapest, Hungary). Data were analyzed using Statistica v. 13 software. Results. In the study group 21 patients with diagnosis DKD+, as compared to 127 subjects withaout DKD–, had longer duration of diabetes [30 (IQR: 21–36) vs. 21 (16–28) years, p = 0.002], higher prevalence of hypertension [14 (67%) vs. 37 (29%), p = 0.002], lower estimated glomerular filtration rate (eGFR) [66 (55–88) vs. 94 (83–106) mL/min/1.73 m2, p < 0.001]. Median MVD compared between groups with and without DKD, was similar for CD34+ vessels/1 mm2 [123 (100–170) vs. 121 (100–170), p = 0.775], CD133+ vessels/1 mm2 [79 (50–100) vs. 79 (63–93), p = 0.823], and for CD31+ vessels/ 1 mm2 [29 (21–46) vs. 38 (17–58), p = 0.454]. Median MVD vWF+ vessels/1 mm2 was lower in the group with than without DKD: 42 (25–54) vs. 54 (43–71), p = 0.009. The values given above were calculated for both layers of the dermis (papillary and reticular dermis). In multivariate logistic regression analysis presence of diabetic kidney disease was associated with lower median vWF+ MVD [odds ratio: 0.97 (95% confidence interval: 0.95–0.99), p = 0.017], with adjustment for age, gender, eGFR value, diabetes duration and presence of hypertension. MVD did not differ significantly between chronic kidney disease stages. Conclusion. In patients with type 1 diabetes and diabetic kidney disease the disturbances in the angiogenesis and vascular maturation are present. The number of mature blood vessels (vWF+) in the skin is reduced. Disturbances in the angiogenesis occur at early stages of diabetic kidney disease

Związek zaburzeń w angiogenezie i dojrzewaniu naczyń w skórze z cukrzycową chorobą nerek w cukrzycy typu 1

Wstęp. Skóra — jako najbardziej dostępny narząd — została wybrana jako model do badania mikrokrążenia i angiogenezy. Celem tego badania była ilościowa i jakościowa analiza związku pomiędzy unaczynieniem skóry (MVD) a obecnością cukrzycowej choroby nerek (DKD) u dorosłych chorych na cukrzycę typu 1 (T1D). Materiał i metody. Badaniem została objęta grupa kolejnych 148 chorych na cukrzycę typu 1 (87 mężczyzn), z medianą wieku 41 lat (IQR: 31–49) i czasem trwania cukrzycy 21 (17–30) lat. Pacjenci byli pod opieką Katedry i Kliniki Chorób Wewnętrznych i Diabetologii Uniwersytetu Medycznego w Poznaniu. Cukrzycową chorobę nerek rozpoznano na podstawie zwiększonej albuminurii u pacjentów z 10-letnim czasem trwania cukrzycy i/lub wykładnikami retinopatii cukrzycowej. Biopsję skóry wykonano na dystalnej części podudzia, przy użyciu jednorazowych 3-milimetrowych sztanc biopsyjnych z suwakiem (Integra™, Miltex®). Do analiz immunohistochemicznych (IHC) wybrano: autoprzeciwciała anty-CD133, anty-CD34, anty-CD31 i anty-vWF. Gęstość naczyń krwionośnych (MVD) wyliczono metodą „gorących miejsc”. Szkiełka skanowano przy użyciu skanera MiraxMidi (Carl Zeiss) i oglądano przy użyciu CaseViewer (3DHISTECH Ltd. Budapeszt, Węgry). Dane analizowano przy użyciu oprogramowania Statistica v. 13. Wyniki. W grupie pacjentów z rozpoznaną DKD (n = 21) w porównaniu z pacjentami bez DKD (n = 127) odnotowano dłuższy czas trwania cukrzycy [30 (IQR: 21–36) vs. 21 (16–28) lat, p = 0,002], częstsze występowanie nadciśnienia tętniczego [14 (67%) vs. 37 (29%), p = 0,002] oraz niższą wartość szacunkowego wskaźnika filtracji kłębuszkowej (eGFR) [66,1 (54,7–87,9) vs. 93,5 (82,5–106,2) ml/min/1,73 m2, p &lt; 0,001]. Mediana MVD naczyń CD34+ na 1 mm2 skóry właściwej wynosiła w grupie DKD+ 123 (100–170), a w grupie DKD– 121 (100–170), p = 0,775; mediana MVD naczyń CD133+ wynosiła odpowiednio 79 (50–100) vs. 79 (63–93), p = 0,823; mediana MVD naczyń CD31+ 29 (21–46) vs. 38 (17–58), p = 0,454; mediana MVD naczyń vWF+ 42 (25–54) vs. 54 (43–71), p = 0,009. Podane wartości obliczono dla obu warstw skóry właściwej (brodawkowatej i siateczkowatej). W modelu wieloczynnikowej regresji logistycznej obecność cukrzycowej choroby nerek była związana z mniejszą MVD dla naczyń krwionośnych vWF+ na 1 mm2 [OR: 0,97 (95% CI: 0,95–0,99), p = 0,017]. Związek ten był niezależny od wieku, płci, wartości eGFR oraz obecności nadciśnienia tętniczego. Grupa badana obejmowała 79 (52,7%) pacjentów z przewlekłą chorobą nerek (CKD) w stadium 1, 58 (39,2%) w stadium 2, 8 pacjentów (5,4%) w stadium 3a i 3 (2%) w stadium 3b. Nie było pacjentów w stadium CKD 4 ani 5. Nie stwierdzono istotnych różnic w MVD w zależności od stadium CKD. Wniosek. U pacjentów z cukrzycą typu 1 i cukrzycową chorobą nerek obserwuje się zaburzenia procesów angiogenezy oraz dojrzewania naczyń. W biopsji skóry obserwowano zmniejszoną liczbę naczyń krwionośnych o wysokiej dojrzałości (vWF+). Zaburzenia angiogenezy w skórze są obecne już we wczesnych stadiach cukrzycowej choroby nerek

Expression Profile of New Gene Markers Involved in Differentiation of Canine Adipose-Derived Stem Cells into Chondrocytes

The interest in stem cell research continuously increased over the last decades, becoming one of the most important trends in the 21st century medicine. Stem cell-based therapies have a potential to become a solution for a range of currently untreatable diseases, such as spinal cord injuries, type I diabetes, Parkinson’s disease, heart disease, stroke, and osteoarthritis. Hence, this study, based on canine material, aims to investigate the molecular basis of adipose-derived stem cell (ASC) differentiation into chondrocytes, to serve as a transcriptomic reference for further research aiming to introduce ASC into treatment of bone and cartilage related diseases, such as osteoarthritis in veterinary medicine. Adipose tissue samples were harvested from a canine specimen subjected to a routine ovariohysterecromy procedure at an associated veterinary clinic. The material was treated for ASC isolation and chondrogenic differentiation. RNA samples were isolated at day 1 of culture, day 30 of culture in unsupplemented culture media, and day 30 of culture in chondrogenic differentiation media. The resulting RNA was analyzed using RNAseq assays, with the results validated by RT-qPCR. Between differentiated chondrocytes, early and late cultures, most up- and down-regulated genes in each comparison were selected for further analysis., there are several genes (e.g., MMP12, MPEG1, CHI3L1, and CD36) that could be identified as new markers of chondrogenesis and the influence of long-term culture conditions on ASCs. The results of the study prove the usefulness of the in vitro culture model, providing further molecular insight into the processes associated with ASC culture and differentiation. Furthermore, the knowledge obtained could be used as a molecular reference for future in vivo and clinical studies

New Molecular Markers Involved in Regulation of Ovarian Granulosa Cell Morphogenesis, Development and Differentiation during Short-Term Primary In Vitro Culture—Transcriptomic and Histochemical Study Based on Ovaries and Individual Separated Follicles

Nowadays, science has a lot of knowledge about the physiology of ovarian processes, especially folliculogenesis, hormone production and ovulation. However, the molecular basis for these processes remains largely undiscovered. The cell layer surrounding the growing oocyte&mdash;granulosa cells&mdash;are characterized by high physiological capabilities (e.g., proliferation, differentiation) and potential for growth in primary cultures, which predisposes them for analysis in the context of possible application of their cultures in advanced methods of assisted reproduction. In this study, we have used standard molecular approaches to analyze markers of these processes in primarily in vitro cultured porcine granulosa, subjected to conditions usually applied to cultures of similar cells. The material for our research came from commercially slaughtered pigs. The cells were obtained by enzymatic digestion of tissues and in vitro culture in appropriate conditions. The obtained genetic material (RNA) was collected at specific time intervals (0 h&mdash;before culture; reference, 48, 98, 144 h) and then analyzed using expression microarrays. Genes that showed a fold change greater than |2| and an adjusted p value lower than 0.05 were described as differentially expressed. Three groups of genes: &ldquo;Cell morphogenesis&rdquo;, &ldquo;cell differentiation&rdquo; and &ldquo;cell development&rdquo; were analyzed. From 265 differently expressed genes that belong to chosen ontology groups we have selected DAPL1, CXCL10, NEBL, IHH, TGFBR3, SCUBE1, DAB1, ITM2A, MCOLN3, IGF1 which are most downregulated and PDPN, CAV1, TMOD1, TAGLN, IGFBP5, ITGB3, LAMB1, FN1, ITGA2, POSTN genes whose expression is upregulated through the time of culture, on which we focused in downstream analysis. The results were also validated using RT-qPCR. The aim of our work was to conduct primary in vitro culture of granulosa cells, as well as to analyze the expression of gene groups in relation to the proliferation of follicular granulosa cells in the model of primary culture in real time. This knowledge should provide us with a molecular insight into the processes occurring during the in vitro cultures of porcine granulosa cells, serving as a basic molecular entry on the extent of the loss of their physiological properties, as well as gain of new, culture-specific traits

Transcriptomic Pattern of Genes Regulating Protein Response and Status of Mitochondrial Activity Are Related to Oocyte Maturational Competence—A Transcriptomic Study

This paper aims to identify and describe new genetic markers involved in the processes of protein expression and modification reflected in the change of mitochondrial activity before and after in vitro maturation of the oocyte. Porcine oocytes collected from the ovaries of slaughtered landrace gilts were subjected to the process of in vitro maturation. Transcriptomic changes in the expression profile of oocyte genes involved in response to hypoxia, the transmembrane protein receptor serine threonine kinase signaling pathway, the &#8220;transforming growth factor &#946; receptor signaling pathway&#8221;, &#8220;response to protein stimulus&#8221;, and &#8220;response to organic substance&#8221; were investigated using microarrays. The expression values of these genes in oocytes was analyzed before (immature) and after (mature) in vitro maturation, with significant differences found. All the significantly altered genes showed downregulation after the maturation process. The most changed genes from these gene ontologies, FOS, ID2, VEGFA, BTG2, CYR61, ESR1, AR, TACR3, CCND2, CHRDL1, were chosen to be further validated, described and related to the literature. Additionally, the mitochondrial activity of the analyzed oocytes was measured using specific dyes. We found that the mitochondrial activity was higher before the maturation process. The analysis of these results and the available literature provides a novel insight on the processes that occur during in vitro oocyte maturation. While this knowledge may prove to be useful in further research of the procedures commonly associated with in vitro fertilization procedures, it serves mostly as a basic reference for further proteomic, in vivo, and clinical studies that are necessary to translate it into practical applications