Endothelial cells were isolated from freshly obtained human umbilical cords by collagenase digestion of the interior of the umbilical vein. The cells were grown in tissue culture as a homogeneous population for periods up to 5 mo and some lines were subcultured for 10 serial passages. During the logarithmic phase of cell growth, cell-doubling time was 92 h. Light, phase contrast, and scanning electron microscopy demonstrated that cultured human endothelial cells grew as monolayers of closely opposed, polygonal large cells whereas both cultured human fibroblasts and human smooth muscle cells grew as overlapping layers of parallel arrays of slender, spindle-shaped cells. By transmission electron microscopy, cultured endothelial cells were seen to contain cytoplasmic inclusions (Weibel-Palade bodies) characteristic of in situ endothelial cells. These inclusions were also found in endothelial cells lining umbilical veins but were not seen in smooth muscle cells or fibroblasts in culture or in situ. Cultured endothelial cells contained abundant quantities of smooth muscle actomyosin. Cultured endothelial cells also contained ABH antigens appropriate to the tissue donor's blood type; these antigens were not detectable on cultured smooth muscle cells or fibroblasts. These studies demonstrate that it is possible to culture morphologically and immunologically identifiable human endothelial cells for periods up to 5 mo.

/pdf/culture-of-human-endothelial-cells-derived-from-umbilical-3vt4ihjkgj.pdf

Culture of Human Endothelial Cells Derived from Umbilical Veins. IDENTIFICATION BY MORPHOLOGIC AND IMMUNOLOGIC CRITERIA

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes.

For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy.

Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

The acute respiratory distress syndrome is a common, devastating clinical syndrome of acute lung injury that affects both medical and surgical patients. Since the last review of this syndrome appeared in the Journal, 1 more uniform definitions have been devised and important advances have occurred in the understanding of the epidemiology, natural history, and pathogenesis of the disease, leading to the design and testing of new treatment strategies. This article provides an overview of the definitions, clinical features, and epidemiology of the acute respiratory distress syndrome and discusses advances in the areas of pathogenesis, resolution, and treatment. Historical Perspective and Definitions . . .

/pdf/the-acute-respiratory-distress-syndrome-4dksnctehq.pdf

The acute respiratory distress syndrome

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-28d1pi1jvo.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy

Allometric scaling relations, including the 3/4 power law for metabolic rates, are characteristic of all organisms and are here derived from a general model that describes how essential materials are transported through space-filling fractal networks of branching tubes. The model assumes that the energy dissipated is minimized and that the terminal tubes do not vary with body size. It provides a complete analysis of scaling relations for mammalian circulatory systems that are in agreement with data. More generally, the model predicts structural and functional properties of vertebrate cardiovascular and respiratory systems, plant vascular systems, insect tracheal tubes, and other distribution networks.

https://science.sciencemag.org/content/sci/276/5309/122.full.pdf

A general model for the origin of allometric scaling laws in biology

Morphometric estimation of pulmonary diffusion capacity: V. Comparative morphometry of alveolar lungs

To determine the extent and sources of adaptive response in gas-exchange to major lung resection during somatic maturation, immature male foxhounds underwent right pneumonectomy (R-Pnx, n = 5) or r...

https://www.physiology.org/doi/pdf/10.1152/jappl.1999.86.4.1301

Compensatory alveolar growth normalizes gas-exchange function in immature dogs after pneumonectomy.

The three-dimensional structure of a composite material found in alveolar exudate of oxygen poisoned lungs but also present in normal lungs is stereologically analysed. It is composed of tubules of 450 A diameter which are tightly packed in a quadratic lattice. The wall of the tub vile is formed by “four-winged” osmiophilic filaments which are located in the corners of the quadratic lattice; their interior is made up of a hydrophilic substance which contains either a tubule or a filament of moderate electron density. The osmiophilic substance of the walls is continuous with associated myelin figures which can be resolved into lamellae with a periodicity of 42 A and can thus be considered to be water crystals of phospholipids. The nature of the content of the tubules, which presumably exerts the formative force on the phospholipid lamellae to form tubules, remains undetermined.

A stereologic electron microscope study of "tubular myelin figures" in alveolar fluids of rat lungs.

Mole rats (Spalax ehrenbergi superspecies) perform the heavy work of digging their subterranean burrows in Israel under highly hypoxic/hypercapnic conditions. Unlike most other mammals, they can achieve high levels of metabolic rate under these conditions, while their metabolic rate at low work rates is depressed. We explored, by comparing mole rats with white rats, whether and how this is related to adaptations in the design of the respiratory system, which determines the transfer of O2 from the lung to muscle mitochondria. At the same body mass, mole rats were found to have a significantly smaller total skeletal muscle mass than ordinary white rats (−22%). In contrast, the fractional volume of muscle mitochondria was larger by 46%. As a consequence, both species had the same total amount of mitochondria and achieved, under normoxia, the same VO2max. Whereas the O2 transport capacity of the blood was not different, we found a larger capillary density (+31%) in the mole rat muscle, resulting in a reduced diffusion distance to mitochondria. The structural pulmonary diffusing capacity for O2 was greater in the mole rat (+44%), thus facilitating O2 uptake in hypoxia. We conclude that structural adaptations in lung and muscle tissue improve O2 diffusion conditions and serve to maintain high metabolic rates in hypoxia but have no consequences for achieving VO2max under normoxic conditions.

https://www.pnas.org/content/pnas/94/5/2062.full.pdf

Working underground: Respiratory adaptations in the blind mole rat

The study of the structural basis of gas exchange function in the lung depends on the availability of quantitative information that concerns the structures establishing contact between the air in the alveoli and the blood in the alveolar capillaries, which can be entered into physiological equations for predicting oxygen uptake. This information is provided by morphometric studies involving stereological methods and allows estimates of the pulmonary diffusing capacity of the human lung that agree, in experimental studies, with the maximal oxygen consumption. The basis for this "machine lung" structure lies in the complex design of the cells building an extensive air-blood barrier with minimal cell mass.

Ewald R. Weibel

Papers

Morphometric estimation of pulmonary diffusion capacity: V. Comparative morphometry of alveolar lungs

Compensatory alveolar growth normalizes gas-exchange function in immature dogs after pneumonectomy.

A stereologic electron microscope study of "tubular myelin figures" in alveolar fluids of rat lungs.

Working underground: Respiratory adaptations in the blind mole rat

Lung morphometry: the link between structure and function.