Endothelial Function - Science topic

Hello

You can consider the following.

1. Thrombo modulin (TM) : It is a membrane protein expressed on the surface of endothelial cells. Soluble TM can be released from injured endothelial cells. It can be measured by ELISA.

2. von Willebrand factor : It is a glycoprotein released from endothelium upon endothelial cell injury. Increased levels are seen in plasma in vascular disease associated with diabetes. It can be estimated by ELISA.

3. E-selectin: It is expressed on the surface of cytokine-activated endothelial cells. Soluble E-selectin released from endothelial cells as a result of shedding of damaged cells. Estimation of this marker can be done by ELISA method.

I hope this is helpful.

Good Luck.

Andy Wijaya Hi Andy, I´ve got fine results with HUVECs by using 10 µg protein/pocket, 10% SDS-Gel. I used eNOS antibody from CellSignaling (D8A6N) #35362 at 1:500 in 5% BSA, incubated overnight @ 4 °C. Secondary antibody was diluted 1:5000.

Good luck!

The method I know is named vascular reactivity, so, I'm sending you the DOI of one of my group paper witch we work with endotelial function and a study material describing it (I'm sorry that the last one is in portuguese).

Doi:

I hope I'm helpful.

Hello

In the American Diabetes Association - 2020 ( https://doi.org/10.2337/dc20-S002 ) says that the diagnosis for diabetes can be given by fasting glucose (> = 126 mg / dL or 7 mM) or by 2 hours of glucose in plasma (> = 200 mg / dL or 11.1 mM) .

In other words, the Krebs solution (11 mM of glucose) would correspond to a glucose load test (ingestion of a glucose load containing the equivalent of 75 g of anhydrous glucose dissolved in water) measured 2 hours after the test.

We could say that the Krebs solution would be a post prandial Krebs

Monika Klimek-Tulwin thanks for your reply.

The insignificant p is for the 3 study with different follow up and population(smaller). however, one study with almost 840 participants showed the positive association but the type of outcome was different in this study. so it makes me think that we cannot completely invalidate the hypothesis. I am now thinking if it could be because of this difference in type of outcome(i.e diabetic measure Fasting plasma glucose, Hba1c & incidence of diabetes). for example, incidence of diabetes is due to many factors like environmental, family history etc.

The reflex here is to put in age as a covariate. The alternative, which makes better use of your age variable, is to use age as the time variable in a survival analysis. Participants enter at the age at which they were first surveyed and exit at the last survey age. This allows you to calculate the effect of your risk factor using a hazard function that starts at the earliest observed entry and ends at the last exit.

Ed Korn has a very good paper on this: Korn EL, Graubard BI, Midthune D. Time-to-event analysis of longitudinal follow-up of a survey: choice of the time-scale. Am J Epidemiol. 1997 Jan 1;145(1):72–80.

1. STORCH, Amanda Sampaio et al. Methods of Endothelial Function Assessment: Description and Applications. Int. J. Cardiovasc. Sci. [online]. 2017, vol.30, n.3 [cited 2018-04-27], pp.262-273.

2. Flammer AJ, Anderson T, Celermajer DS, et al. The Assessment of Endothelial Function – From Research into Clinical Practice. Circulation. 2012;126(6):753-767.

3. D Tousoulis, C Antoniades, and C Stefanadis.Evaluating endothelial function in humans: a guide to invasive and non-invasive techniques. Heart. 2005 Apr; 91(4): 553–558

Greetings Yonghui,

I worked with HUVEC throughout graduate school and regularly verifed enhanced expression of ICAM-1 or VCAM-1 in the presence of TNF (10ng/ml) after 16-20hrs exposure compared with untreated controls. Immunocytochemistry with a primary and secondary antibody was used. ICAM-1 showed a very large (4-20fold) higher signal which was obvious by eye under the microscope. VCAM-1 also showed an increased signal, but the effect was generally observed in a smaller percentage of the cell population, and the signal was lower in magnitude relative to ICAM-1. I hope this is useful for you. In addition, I am a bit surprised your cells are still alive after 48hrs treatment with 10ng/ml tnfa. In my experience the cells were often in bad shape if 20hrs exposure was exceeded. I had cultured the cells in M199 with 10%fbs and other supplements ( lglut, ecgs, pen strep). Since you are confident your TNFa stock is active, perhaps consider the staining method (are you getting backround signal in both groups, perhaps?). If you think the control/no treatment group is being inflamed as well, then I'd expect it to be a very large signal if it is equal to the tnfa treated group. If this is not the case then perhaps your tnfa-treated cells are dying/dead, or the surface ICAM-1 was primarily cleaved at that time point (48hrs). 

You have good reference articles (Cheng and Reneman). Physic at the microcirculation level has nothing to do with what is seen in big vessels (limit 250 micrometers). This is the border. None of the laws that applies over this limit applies under the limit. It's very tricky. With strange things and their consequences, for example the notion of additional drop pressure, depending on how much red cells flow in a small capillary and if they are separated or assembled...

I think EDHF is a concept EDH is more accurate as it describes a set of processes  which evoke  endothelium dependent hyperpolarization and relaxation of smooth muscle. These include factors but also gap juntional communication. I Think most agree the term EDHF is redundant unless referring to a specific factor like an EET and then it is probably  better to just refer to that factor. Indeed by this definition  NO is also and EDHF and I think no one uses the term EDRF anymore and just refer to NO. IF you have relaxation you can't attitude to a  a single factor or is independent  of  a factor  then EDH  is probably  the correct term.

Hello. When you isolate microsomes, depending on the method, you may have several organelles. For example, it is easy to co-fractionate mitochondria and ER. You should always use bonafide organelle markers to learn the identity of your microsomes. Depending on the desired separation, whatever you get may be fine as long as you don't overstate your results. Hope this helps.

The authors of the paper mentioned by the OP used Matrigel, type I collagen and fibronectin as substrates for BAEC culture.  Matrigel is a typical surrogate for basement membrane, while type I collagen and Fn are components of interstitial extracellular matrix, which the BAEC might be expected to see if the basement membrane was disrupted by inflammation or disease.  This kind of approach of varying the substrate matrix and monitoring cellular response is fairly typical, and has been used for some time, e.g. Madri, Pratt, & Yannariello-Brown. Amer. J. Pathol. 132:18, 1988.

OK, found it now, the following is extracted from the methods section of :

ISOLATED PERFUSED RABBIT CORONARY ARTERY AND AORTIC STRIP PREPARATIONS: THE ROLE OF ENDOTHELIUM-DERIVED RELAXANT FACTOR

BY T. M. GRIFFITH, A. H. HENDERSON, D. HUGHES EDWARDS AND M. J. LEWIS, J. Physiol. 351, 13-24, 1984.

Endothelial histology: Arterial preparations at the end of an experiment were stained by a modification of the method

of Poole, Sanders & Florey (1958). Perfused coronary artery preparations were perfused intraluminally with1% glutaraldehyde for 5min followed by1% silver nitrate for 2 min and then by a solution containing 3% cobalt bromide and 3% ammonium bromide for 2min. It was necessary to perfuse the arteries with glutaraldehyde before the silver nitrate in order to fix the tissue and prevent the severe constriction which the silver nitrate commonly caused and which itself caused further loss of endothelium. For the aortic strips the staining procedure was similar except that the glutaraldehyde step was omitted.

The endothelial surface of the arteries was visualized with a binocular microscope. An example of a coronary artery partially denuded of endothelium is shown in P1. 1. The entire surface of each preparation examined was scanned and a visual estimate made of the percentage of the surface stilcoveredbyendothelium.

Hope that helps.

Dear. Sneha Agarwal

thanks for your good advise.

We used to call this spontaneous activity and Dr Ward is absolutely right; it is really not a problem as long as you are able to quantify changes in vessel tone.

I remember on the other hand that mesenteric arteries (rat, rabbit, mouse, dog etc) actually show this type of 'behaviour' even at basal tone (i.e. without being pre-contracted with NE, Ang II or KCl).

Are these helicoidal strips and which physiological buffer are you using? In addition, if  you want to reduce these oscillations at least with an alpha agonist, try methoxamine instead and add indomethacin in your assay buffer. The addition of an inexpensive betablocker may also help.

Good luck

most stimple is cytokine beads array using flow cytometry

I'm sorry if i didn't get you right, but what exactly is ethically unaccepted?

The best method depends on which patient collective you want to measure and to which extend you want to or can use provocation tests.

On of the classic tests would be the patients reaction regrading post-occlusive reactive hyperaemia. You can measure the response by device like the O2C by LEA or have a look at the Periflux devices. The choice also depends on your potential needs for further possibilities like trans-dermal drug application etc.

You can also remove the endothelial layer mechanically with e.g. a hair by rubbing the lumen gently. Afterwards you can check the vasoconstrictor capacity with high concentration potassium (e.g. KPSS 60 mM) and the endothelium dependent relaxation with acetylcholine/carbachol after pre-constriction with e.g. 30 mM KPSS. Good luck

I use one of my own hairs!... I do it while the artery is mounted between wires with some tension on and gently rub in a circular motion with very gentle pressue against the artery wall/endothelium in one direction....this decreases Ach responses to less then 10%.

Arun

A widespread approach is the use of hyperglycemia in vitro. Most groups use 5 mM glucose for control and 25 mM for hyperglycemia, mostly for 48 h or more. Please make sure to correct for the elevated osmolality by adding mannitose to the control culture. Good luck! JW

Thank you. I will check it out!

Hypoxia is a relative term. In a "normoxic" organism breathing air, many organs are "hypoxic". Moreover, ischemia implies local (not always systemic) hypoxia but the reverse is not rue. So, there is no established marker of hypoxia, but many markers of the response to hypoxia are available: most if not all of them have been listed above. So my suggestion would be to use the marker related to the "hypoxic" response you are investigating in your specific research. Good luck!

Dear Alex, many answers on your question you could find in Circulation (very useful journal for vascular research). Please note the influence of sympatic nerv.system in combination with NO in your case on endothelial function. I also could advise you to read science literature review from ITAMAR (endo PAT manufacture).

I have another question for you. How did you make fixation of ultrasound probe in your case? This detail could influence on final result of measurement.

Regards