On the basis of the seminars and additional reading,
students should be able to:
1. Use the Starling Equation to calculate transient and steady state changes in filtration rate after physiological and pathological alterations in vascular and interstitial hydrostatic and oncotic pressures, blood flow and permeability.
2. Determine the relative contributions of diffusive and convective fluxes to transport of different sized molecules from blood to tissue
3. Discern the differences in physiological outcomes between haemodynamic and permeability changes during inflammation and angiogenesis.
4. Be able to explain the most important molecular and cellular processes that occur during changes in permeability, and during angiogenesis
LECTURE 1. Fluid and solute exchange
Important references are in bold. Michel's NIPS Paper is extremely useful reading for this lecture. Most of the papers quoted during the lecture are review articles and easily understood. The research papers are more difficult, but the Michel and Phillips paper is critical. The Renkin et al paper is a classic. Curry's Handbook of Physiology paper is too difficult unless you really love maths. The following chapter (Michel's) makes much easier reading.
Background
In 1896 Ernest Starling described the forces regulating fluid exchange between the blood and the tissue spaces. These are currently described in the equation:
Jv=Lp.A.[(Pc-Pi)-s(Pp-Pi)]
Starling Equation
After making measurements of human capillary pressure at arterial, middle and venous ends of the capillary, and human plasma colloid osmotic pressure, Eugene Landis used this relationship in 1963 to propose that fluid filters out of the capillary at the arterial end and is absorbed at the venous end.
The filtration rate (Jv) and the rate of solute flux (Js) controls the interstitial protein concentration and hence the interstitial oncotic pressure.
Ci=Js/Jv
Solute flux occurs by diffusion and convection.
Diffusive solute flux occurs according to Fick's Law
Js (diff) = PA(Cp-Ci)
Convective solute flux is by Solvent Drag
Js (conv) = Jv(1-s)Cp
Total solute flux is the sum of the two.
Conversely, as filtration rate falls, so the interstitial protein concentration rises towards the plasma protein concentration, so restoring the filtration rate.
Sustained absorption from the venous end of a capillary is therefore not usually the case. This was shown by Michel and Phillips in 1987 using the Landis isolated perfused capillary preparation.
The problems with the Landis model of filtration absorption stemmed from unrepresentative capillary pressure measurements, under-estimation of the contribution of interstitial forces, and the under-estimation of lymph flow.
Problems
a) Calculate Jv (in ml.min-1.100g tissue-1) for typical values given.
b) Extrapolate this to the whole body of a 70kg person. What proportion of plasma volume turns over each day?
c) Calculate lymph flow for the tissue and for the whole body, assuming a steady state (no swelling).
Lp=0.16x10-7 cm.sec-1.mmHg-1, surface area is 100cm2g-1. Capillary pressure=20mmHg, Interstitial hydrostatic pressure=0, plasma colloid osmotic pressure=25mmHg, interstitial oncotic pressure=5mmHg, oncotic reflection coefficient=0.95, haematocrit is 45%
d) Plot the pressure filtration rate curve you would expect from a single capillary perfusion experiment where the extravascular oncotic pressure is set at 25mmHg.
Capillary Permeability and Inflammation.
Important references are in italics. Michel and Curry's Physiological Review paper has an excellent description of endothelial cell behaviour during inflammation. Copies of the Neal, MacDonald, Feng and Bates Microcirc reviews are in my office. Adamson's paper is a classic, as is Bundgaard's (in my office).
Endothelial cells line the vessel wall and form a tight overlapping barrier to macromolecules. The endothelial junctions are the primary route for hydrophilic solutes to move across the endothelial barrier. These junctions - or clefts - are interspersed with tight junctions, which act as barriers to solute flux. The tight junctions are discontinuous and solutes may pass around the discontinuities. These clefts form a barrier to protein flux, so that they have a high oncoticreflection coefficient to solutes such as albumin (0.9-0.99). As fluid filters out of these clefts the tissue side of the cleft is therefore exposed to dilute filtrate at a protein concentration of (1-s)Cp. If capillary pressure falls, then the filtration rate falls, until the rate of diffusion from both the plasma protein through the cleft and the interstitial protein back into the cleft, restores the oncotic pressure difference, and filtration is restored.
Permeability of the vessel wall can be described by three coefficients, Solute permeability (Ps), hydraulic conductivity (Lp) and oncotic reflection coefficient (s). Solute permeability is defined by Fick's law, and is controlled by the restricted diffusion coefficient of the solute, the area of the pores relative to the area of the vessel, the number of pores and their length, and the solute size. Larger solutes have a lower permeability which is described well for small molecules (up to albumin) by a 5nm pore, and for larger molecules by a very small number of very large pores. The reflection coefficient depends on the pore size and the solute size, and increases with increasing pore size. Hydraulic conductivity is controlled by the pore radius, pore length and relative viscosity in the cleft.
Permeability can be measured by measuring solute flux, or fluid flux under known concentration or pressure differences. Alternatively, reflection coefficient can be measured by determining the minimum interstitial to plasma protein concentration.
Inflammation.
Endothelial cells can be activated to open channels for water and solute flux. This occurs by inflammatory mediators such as bradykinin, histamine, etc, and angiogenic factors such as vascular endothelial growth factor. They do this by increasing intracellular calcium and so stimulating cytoskeletal rearrangements which lead to:
Transcellular Gaps , Intercellular Gaps , and Vesiculovacuolar organelles
Questions
1. Calculate the diffusive and convective fluxes of glucose and albumin. Which flux is more important for delivery of each solute to the tissue? Would you expect P, A, Cp or Ct to vary between tissues
PAglucose=0.08ml/sec/100g, PAalbumin=1.2x10-4ml/min/100g, Cp=60g/l for albumin, 5mM for glucose, Ct=4.7mM for glucose, and 10mg/ml for albumin, s=0.1, and MW= 180 for glucose.
6. What happens to (up, down or no change) the following parameters for albumin and water during the following states. Please state whether you mean acutely (within a few minutes) or chronically (over five minutes to an hour). Which parameters change due directly to the primary challenge to the body, and which ones change as a response to the primary challenge.
Jv Js Pc Pi 1c 1i Cp Ct P Lps A JL
Heavy exercise
Hot weather
Severe haemorrhage
Sprained ankle
Burned Finger
Filariasis*
Deep venous thrombosis
*Filariasis is a parasitic infection of the lymph nodes which results in lymphatic insufficiency.
Angiogenesis
Angiogenesis is the formation of new blood vessels from existing vasculature. It is necessary for normal tissue function during development, and modulation of function, e.g. in skeletal muscle during training. It is also critical for endometrial formation and corpus luteum development. It is strongly stimulated in tumours, arthritis, psoriasis, and during wound healing, including in myocardial infarction. Angiogenesis can be measured in vitro and in vivo by a variety of mnodels. Angiogenesis is stimulated by production of vascular growth factors in response to hypoxia, or constitutive upregulation in tumours. Up regulation of Vascular endothelial growth factors can be brought about at the transcriptional and translational level, since VEGFs have an Internal Ribosome Entry Site. VEGFs cause an increase in vascular permeability, stimulation of release of enzymes to break down connective tissue, endothelial cell migration and mitosis, detachment of pericytes and smooth muscle cells, and invasion of connective tissue by the endothelial cells all of which occurs during sprouting. Once another growing tip is connected the developing lumens connect and blood flow occurs through the new capillary loop. This results in production of angiopoetins, which attract pericytes, stimulate collagen and basement membrane synthesis, and smooth muscle cell attraction to stabilise the vessels.
Problems:
Under what conditions would angiogenesis occur in normal adult human beings?
In what diseases might you expect angiogenesis to be a contributing factor?
REFERENCES