Birth weight and characteristics of endothelial and smooth muscle cell cultures from human umbilical cord vessels.

BACKGROUND: Low birth weight has been related to an increased risk for developing high blood pressure in adult life. The molecular and cellular analysis of umbilical cord artery and vein may provide information about the early vascular characteristics of an individual. We have assessed several phenotype characteristics of the four vascular cell types derived from human umbilical cords of newborns with different birth weight. Further follow-up studies could show the association of those vascular properties with infancy and adulthood blood pressure. METHODS: Endothelial and smooth muscle cell cultures were obtained from umbilical cords from two groups of newborns of birth weight less than 2.8 kg or higher than 3.5 kg. The expression of specific endothelial cell markers (von Willebrand factor, CD31, and the binding and internalization of acetylated low-density lipoprotein) and the smooth muscle cell specific alpha-actin have been evaluated. Cell culture viability, proliferation kinetic, growth fraction (expression of Ki67) and percentage of senescent cells (detection of beta-galactosidase activity at pH 6.0) have been determined. Endothelial cell projection area was determined by morphometric analysis of cell cultures after CD31 immunodetection. RESULTS: The highest variation was found in cell density at the confluence of endothelial cell cultures derived from umbilical cord arteries (66,789 +/- 5,093 cells/cm(2) vs. 45,630 +/- 11,927 cells/cm(2), p < 0.05). Morphometric analysis indicated that the projection area of the artery endothelial cells (1,161 +/- 198 and 1,544 +/- 472 microm(2), p < 0.05), but not those derived from the vein from individuals with a birth weight lower than 2.8 kg was lower than that of cells from individuals with a birth weight higher than 3.5 kg. CONCLUSION: The analysis of umbilical cord artery endothelial cells, which demonstrated differences in cell size related to birth weight, can provide hints about the cellular and molecular links between lower birth weight and increased adult high blood pressure risk.

Procedure to consistently obtain endothelial and smooth muscle cell cultures from umbilical cord vessels.

The prenatal history of an individual can be responsible to some extent for the occurrence of several diseases later in life. Thus, low birth weight has been related to an increased risk of developing hypertension or type 2 diabetes. The molecular and cellular basis of this increased risk could be found in body fluids and cell types that can be obtained just after birth. To get this unique information, a methodology was developed to consistently obtain cultures of 4 cell types, endothelial and smooth muscle cells from both the vein and the arteries present in the umbilical cord of an individual. From 21 umbilical cords processed, 82 of the 84 possible cell cultures were obtained. The cell cultures exhibit the expected cell morphology and cellular characteristics. Thus, endothelial cells express the von Willebrand factor, CD31, as well as bind and internalize acetylated low-density lipoprotein. Vascular smooth muscle cells express the distinctive alpha-actin. Cell cultures can be cryopreserved and grow healthy for several passages. No influence of birth weight of the newborn has been found in the time required to obtain a primary cell culture for any of the 4 cell types. In conclusion, the procedure developed allows one to routinely obtain actively growing vascular cell cultures that could be used to study the molecular and cellular basis of vascular diseases that emerge in adulthood.

First-year blood pressure increase steepest in low birthweight newborns.

AIM: The present research has been undertaken prospectively to study the impact of birthweight and growth pattern on blood pressure changes from birth through the first year of life. METHODS: Parents of newborns born at term (gestational age > 37 weeks) after uncomplicated pregnancies and in the absence of perinatal illness were randomly invited to allow their children to participate in the study. One hundred and forty-nine (84 male and 65 female) newborns were included in the present analysis. The newborns were divided into four groups according to birthweight: < 2500 g (n = 23); 2500-2999 g (n = 39); 3000-3500 g (n = 48); and > 3500 g (n = 39). RESULTS: At birth systolic and diastolic blood pressure were significantly lower and heart rate was significantly higher in those children with the lowest birthweight as compared to those in the other groups. During the first month of life a significant trend, inversely related to birthweight, was present for systolic as well as diastolic blood pressure. After the first month of life, at 3, 6, 9 and at 12 months, systolic and diastolic blood pressure were similar across birthweight groups. In a multiple regression analysis, birthweight was a positive independent determinant of systolic blood pressure at birth and an inverse independent determinant of the increment of systolic blood pressure during the first month of life and of the systolic blood pressure at the end of the first year. CONCLUSIONS: In summary, the present study goes further towards understanding blood pressure changes in low birthweight babies. Beginning at birth, both blood pressure values, as well as changes in blood pressure, provide information about the impact of intrauterine life on the risk of developing hypertension later in life.

A single point mutation in the low-density lipoprotein receptor switches the degradation of its mature protein from the proteasome to the lysosome.

Pathogenic mutations in the low-density lipoprotein receptor prevent cholesterol uptake and cause familial hypercholesterolemia. In comparison to the biogenesis and endocytic trafficking of this receptor and some of its mutants, their degradation mechanisms are not well understood. Therefore, to gain some insights into this aspect, we analyzed the effects of proteasomal and lysosomal inhibitors on the levels of the wild type low-density lipoprotein receptor and a mutant form, C358Y, which was prevalent in a sample of Spanish familial hypercholesterolemia patients. In transfected cells, the mutant C358Y exhibited lower activity than the wild type receptor, as well as retarded post-translational processing of its precursor to the mature form. Interestingly, about 30% of the mutant precursor was degraded by a lysosomal pathway. Moreover, its mature form was more rapidly degraded than the wild type receptor (half lives of 5.3 and 10.9 h, respectively) and its degradation was exclusively dependent on a lysosomal pathway. In contrast, the mature form of the wild type receptor was mainly degraded by proteasomes and, to a minor extent (30%), by lysosomes. We conclude that a single mutation in the low-density lipoprotein receptor switches the degradation of the mature receptor from a proteasomal to a lysosomal pathway which degrades the protein at a faster rate. This suggests cooperation of proteasomes and lysosomes in the degradation of the low-density lipoprotein receptor and adds an intriguing new aspect to our understanding of receptor-mediated endocytosis.

Disturbed cholesterol traffic but normal proteolytic function in LAMP-1/LAMP-2 double-deficient fibroblasts.

Mice double deficient in LAMP-1 and -2 were generated. The embryos died between embryonic days 14.5 and 16.5. An accumulation of autophagic vacuoles was detected in many tissues including endothelial cells and Schwann cells. Fibroblast cell lines derived from the double-deficient embryos accumulated autophagic vacuoles and the autophagy protein LC3II after amino acid starvation. Lysosomal vesicles were larger and more peripherally distributed and showed a lower specific density in Percoll gradients in double deficient when compared with control cells. Lysosomal enzyme activities, cathepsin D processing and mannose-6-phosphate receptor expression levels were not affected by the deficiency of both LAMPs. Surprisingly, LAMP-1 and -2 deficiencies did not affect long-lived protein degradation rates, including proteolysis due to chaperone-mediated autophagy. The LAMP-1/2 double-deficient cells and, to a lesser extent, LAMP-2 single-deficient cells showed an accumulation of unesterified cholesterol in endo/lysosomal, rab7, and NPC1 positive compartments as well as reduced amounts of lipid droplets. The cholesterol accumulation in LAMP-1/2 double-deficient cells could be rescued by overexpression of murine LAMP-2a, but not by LAMP-1, highlighting the more prominent role of LAMP-2. Taken together these findings indicate partially overlapping functions for LAMP-1 and -2 in lysosome biogenesis, autophagy, and cholesterol homeostasis.

Changes in the proteolytic activities of proteasomes and lysosomes in human fibroblasts produced by serum withdrawal, amino-acid deprivation and confluent conditions.

The contribution of the main proteolytic pathways to the degradation of long-lived proteins in human fibroblasts grown under different conditions was investigated. The effects of various commonly used pharmacological inhibitors of protein degradation were first analysed in detail. By choosing specific inhibitors of lysosomes and proteasomes, it was observed that together both pathways accounted for 80% or more of the degradation of cell proteins. With lysosomal inhibitors, it was found that serum withdrawal or amino-acid deprivation strongly stimulated macroautophagy but not other lysosomal pathways, whereas confluent conditions had no effect on macroautophagy and slightly activated other lysosomal pathways. Prolonged (24 h) serum starvation of confluent cultures strongly decreased the macroautophagic pathway, whereas the activity of other lysosomal pathways increased. These changes correlated with electron microscopic observations and morphometric measurements of lysosomes. With proteasomal inhibitors, it was found that, in exponentially growing cells in the absence of serum, activity of the ubiquitin-proteasome pathway increases, whereas under confluent conditions the contribution (in percentage) of proteasomes to degradation decreases, especially in cells deprived of amino acids. Interestingly, in confluent cells, the levels of two components of the 19 S regulatory complex and those of an interchangeable beta-subunit decreased. This was associated with a marked increase in the levels of components of PA28-immunoproteasomes. Thus confluent conditions affect proteasomes in a way that resembles treatment with interferon-gamma. Altogether, these results show that the activity of the various proteolytic pathways depends on the growth conditions of cells and will be useful for investigation of the specific signals that control their activity.

Role of proteasomes in the degradation of short-lived proteins in human fibroblasts under various growth conditions.

Degradation of proteins in the cells occurs by proteasomes, lysosomes and other cytosolic and organellar proteases. It is believed that proteasomes constitute the major proteolytic pathway under most conditions, especially when degrading abnormal and other short-lived proteins. However, no systematic analysis of their role in the overall degradation of truly short-lived cell proteins has been carried out. Here, the degradation of short-labelled proteins was examined in human fibroblasts by release of trichloroacetic acid-soluble radioactivity. The kinetics of degradation was decomposed into two, corresponding to short- and long-lived proteins, and the effect of proteasomal and lysosomal inhibitors on their degradation, under various growth conditions, was separately investigated. From the degradation kinetics of proteins labelled for various pulse times it can be estimated that about 30% of newly synthesised proteins are degraded with a half-life of approximately 1h. These rapidly degraded proteins should mostly include defective ribosomal products. Deprivation of serum and confluent conditions increased the degradation of the pool of long-lived proteins in fibroblasts without affecting, or affecting to a lesser extent, the degradation of the pool of short-lived proteins. Inhibitors of proteasomes and of lysosomes prevented more than 80% of the degradation of short-lived proteins. It is concluded that, although proteasomes are responsible of about 40-60% of the degradation of short-lived proteins in normal human fibroblasts, lysosomes have also an important participation in the degradation of these proteins. Moreover, in confluent fibroblasts under serum deprivation, lysosomal pathways become even more important than proteasomes in the degradation of short-lived proteins.