MICROVASCULARRESEARCH23,392-398 (1982)
Abstracts of Papers Presented at a Meeting of the British Microcirculation Society September 10 and 11, 1981, St. Aidan's College, University of Durham, Durham, England 1. Angiogenesis Factors and Their Inhibitors. S. KUMAR,Christie Hospital, Manchester M20 9BX, England.
Following the first demonstration a decade ago of angiogenic activity in semi-purified extracts of tumours by Folkman and his associates (1971, J. Exp. Med. 133, 275) a number of other investigators have also found such an activity in tumours and nontumour sources (Auerbach, 1981, In "Lymphokines" (E. Pick, ed.), Academic Press, New York). Neither the chemical nature nor the relationship of the angiogenesis factors is known. This is largely the result of the lack of a quantitative assay and of the fact that only exceedingly, small quantities of angiogenic factors can be purified. At Manchester, using an in vivo chorioallantoic membrane assay (CAM), we have isolated a lowmolecular-weight ( - 300 daltons) angiogenesis factor(s) from animal and human tumours, regenerating rat liver, human synovial fluid, human myocardial infarct, and cat retina. Purified tumour-derived material in vitro induces stimulation of capillary endothelial cells (Schor et al., 1980, Br. J. Cancer 41, 970), but has no mitogenic effect on endothelial cells obtained from large blood vessels (Briedey et al., unpublished data). In preliminary studies, Brierley has also found that although low-molecularweight angiogenesis factor from tumour and cat retina induces in vivo stimulation of angiogenesis in CAM, it is only tumour angiogenesis factor which enhances proliferation of capillary endothelial cells in vitro,. This may reflect an important difference in angiogenesis factors derived from tumours and normal tissue. The mechanism of action of angiogenesis factors has not been investigated to any extent, although it is thought that activation of latent metalloproteinases is involved. Inhibitors of angiogenesis when they can be identified would be important in prevention of vascular proliferation. Maugh has written a lucid review of methods preventing blood vessel growth in cancer and other diseases wherein excessive proliferation of blood vessels is a feature (1981, Science 212, 1374).
2. Neovascularisation in the Chick Chorioallantoic Membrane. R. A. FRASER,G. J. HOOD, AND J. G. SIMPSON, Department of Pathology, University of Aberdeen, Aberdeen AB9 2ZD, Scotland.
Tumour angiogenesis factor and certain vasoactive agents (histamine, 5-hydroxytryptamine, and adenosine diphosphate) produce neovascularisation when applied to the chick chorioallantoic membrane (Fraser et al., 1979, Microvasc. Res. 18, 285). These vasoactive agents, but not tumour angiogenesis factor, are also capable of increasing the growth rate of cultured human endothelial cells (Fraser and Stalker, 1980, Bibl. Anat. 20, 633-636). Since these agents are present in high concentrations in mast cells, it is possible that mast cells are involved in the in vivo mediation of the effects of tumour angiogenesis factor. A light microscopic comparison has been made of the effects of tumour angiogenesis factor and 5-hydroxytryptamine (5HT) on the chick chorioallantoic membrane. Turnout angiogenesis factor or 5HT, in solution in phosphate-buffered saline, was applied to chick chorioallantoic membranes on the tenth day of egg incubation. Five days later, the membranes were fixed, removed, and examined microscopically. Both tumour angiogenesis factor and 5HT induced altered vascular growth patterns and neovascularisation. Each caused the same degree of vascular reaction. The main difference was seen in the mast cell content. Tumour angiogenesis 392 0026-2862/82/030392-07502.00/0 Copyright © 1982by AcademicPress, Inc. All rightsof reproductionin any form reserved. Printed in U.S.A.
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factor, but not 5HT, caused a massive increase in mast cell numbers at the affected sites. In neither case did epithelial hyperplasia occur. The association of mast cell hyperplasia with the neovascularisation found after tumour angiogenesis factor supports the hypothesis that mast cells are involved in the mediation of the effects of tumour angiogenesis factor on the microcirculation.
3. Physical Influences Involved in Angiogenesis. H. Inhibition of Epithelial Syncretic Activity. R. L. BARNHILLArid T. J. RYAN, Department of Dermatology, University of Oxford, Slade Hospital, Oxford, OX3 7JH, England. Perturbed epithelium may exert mechanical effects on surrounding vasculature, orienting and drawing in vascular and fibre networks (Barnhill and Ryan, 1981, Microvasc. Res. 22, 233). One or more mechanisms could account for this syncretic activity: (1) cellular proliferation and/or buckling of an epithelial layer, (2) cellular locomotion, (3) cellular contractility, or (4) drying effects. We examined the effects of a number of agents on epithelial contraction stimulated by 4-mm fabric discs applied to the chorioallantoic membrane of the chicken embryo. The following agents were applied daily: hydrocortisone 10 -3 M, methotrexate 2.5 mg/ml, arachis oil, cytochalasin E 20 ixg/ml, acetylsalicylic acid 10 -3 M, EDTA 10 3 M, and 7-aminobutyric acid 10 -3 M. Responses were recorded after 4 days and compared to the effects of these agents on neovascular responses to 2-mm Millipore filter discs (Barnhill and Ryan, 1981, J. Invest. Dermatol. 76, 428). The contraction was inhibited and neovascularization significantly decreased by hydrocortisone, methotrexate, and arachis oil. The other compounds produced no significant effect. These findings demonstrate that hyperplasia is the predominant mechanism in the chick chorioallantoic membrane accounting for this physical effect. This does not mean that other processes such as fibroblast contractility or drying are not important in other situations such as wound healing. Thus epithelial proliferation may be one of several factors having important physical influence on ensuring an adequate vascular supply. Supported by the Psoriasis Association of Great Britain.
4. The Possible Role of the Microcirculation in Regulating Compensatory Hyperplasia. J. D. S1MYET'r, University of Newcastle upon Tyne, Department of Pathology, Royal Victoria Infirmary, Newcastle upon Tyne NE1 4LP, England. In many tissues removal or damage causes an increased mitotic rate in the remaining functional cells ("compensatory hyperplasia"). To account for the apparent tissue specificity of this reaction most theories assume the existence of tissue-specific chemical regulators. Examples of such putative control factors are the "chalones" (inhibitors of mitosis), "wound hormones" (stimulators of mitosis), and increased levels of stimulatory metabolites resulting from "functional overload" of the undamaged tissue. When ectopic grafts of kidney (Simnett et al., 1977, Anat. Rec. 187, 273-280) or liver (Simnett et al., 1977, Experientia 33, 1457-1458) were placed in the lung of Xenopus frogs subsequent removal of the contralateral lung lead to compensatory hyperplasia in the remaining lung and also in the ectopic grafts. In this case the response was not truly tissue specific, but more correctly "location specific." Ectopic grafts shared a common vascular supply with the surrounding lung and it was proposed that compensatory hyperplasia is caused by the increased blood flow which is a typical feature of tissue damage and which, in turn, is mediated by the autonomic innervation. Previous reports (see Goss, 1964, "Adaptive Growth," Academic Press, New York/London) that denervation of the salivary gland prevents compensatory hyperplasia are consistent with this hypothesis.
5. The Effect of Parasympathetic Nerve Stimulation of the Albumin Permeability of Rabbit Salivary Gland Capillaries. P. D. SPENCER, L. H. SMAJE,W. P. PENN, AND S. LAM, Department of Physiology, Charing Cross Hospital Medical School, London W6 8RF, England. The fenestrated capillaries of salivary glands are highly permeable to small solutes but this is not increased by parasympathetic nerve stimulation (Mann et al., 1979, J. Physiol. 297, 355-367). The
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present experiments were designed to study the effect of parasympathetic stimulation on macromolecular permeability. The accumulation of radiolabelled human serum albumin in rabbit submandibular salivary glands after a single intravenous injection was used to estimate albumin permeability. A mathematical model, modified from that of Johnson (1966, Amer. J. Physiol. 211, 1216-1263) to include solvent drag effects and the disappearance of albumin from the plasma, was devised to help interpret the results. In paired experiments, the plasma-equivalent albumin space rose by a mean of 1.2 ml/100 g at 60 rain from an initial value of 7.5 ml/100 g (assumed to be equal to the intravascular space) and by 7.1 ml/100 g after 4-6 hr. The extravascular, extraceUular space, as measured by 5~Crlabelled EDTA was 32.9 ml/100 g. After 60 min stimulation the albumin space rose by a further 1.2 ml/100 g over the control value. Our analysis of these results suggests that: (1) The permeability-surface area product (PS) for albumin is less than 0.003 ml/min/100 g, similar to the low value obtained by Koo et al. (1980), Microvaac. Res. 20, 256-257) who used an entirely different method. (2) The small increase in albumin transport obtained in stimulated glands can be accounted for by an increase in surface area and capillary pressure due to the accompanying vasodilatation, rather than by an alteration in the permeability properties of the vessel wall.
6. Measurements o f Myocardial Capillary Permeability in the Perfused Rabbit Heart: A Review o f the Effects o f Flow Rate, Adenosine, ATP, and Albumin. G. E. MANN, Department of Physiology, Queen Elizabeth College, University of London, London W8 7AH, England. Myocardial capillary permeability to small hydrophilic solutes has been investigated extensively using the single-passage, multiple-tracer dilution technique. In the present study isolated rabbit hearts were perfused at flow rates between 0.2 and 5 ml/min • g with a Krebs solution to which unlabelled cyanocobalamin, insulin, and albumin were added. Following a close-arterial injection (100 ~1 in 1-2 sec) of an intravascular reference tracer (~25I-or t3q-serum albumin) and three test tracers (22Na, 5~CrEDTA, [SYCo]cyanocobalamin, or t25I-insulin) into the perfusate supplying the coronary arteries, the sinus effluent was immediately sampled at 0.3 to l-sec intervals. The initial capillary extraction (E) was determined from the dilution curves using the expression E = 1 - CJCr, where C, and Cr are the concentrations of the test and reference tracers, respectively. Capillary permeability-surface area products (PS, ml/min • g) were estimated from PS = - F • In (1 - E) and a brief review of this analysis has been published (Mann, 1979, J. Physiol. 291, 7-8P). PS values for 22Na increased continuously with increments in flow, whereas PS for ~Cr-EDTA, [57Co]cyanocobalamin, and ~25Iinsulin reached constant values at higher perfusion rates (Mann, 1981, J. Physiol. 319). These findings are in agreement with the original observations of Alvarez and Yudilevich (1969, J. Physiol. 202, 45) in the dog heart in situ. In the presence of either adenosine or ATP (l mmole/liter) significant increases in PS were observed, however, this effect was most pronounced at flow rates below 1 ml/ rain • g (22Na, 280%; EDTA, 210%; cyanocobalamin, 88%). Since permeability ratios were not altered significantly, these results suggest a recruitment of capillary surface area in the presence of the vasodilators. When hearts were initially perfused with an albumin-free solution and then with albumin (10 g/liter), PS for all test molecules decreased significantly (Mann, 1981). As proposed by Curry and Michel (1980, Microvasc. Res. 20, 96) albumin may decrease capillary permeability by interacting with the network of fibrous molecules lining the transcapillary pathways.
7. The Use o f Histochemical Techniques to Search for Periarteriolar Nerves in Human Breast Tissue. J. E. KENDALL AND C. J. JONES, Department of Zoology, University of Durham, Science Laboratories, South Road, Durham DHI 3LE, England. Ultrastructural studies of arterioles in biopsy material from human breast (21 arterioles, 7 patients) have failed to reveal any surrounding nerves (Jones and Kendall, 1981, Microvasc. Res. 22, 235). This may not mean that the vessels lack a nerve supply, simply that the nerves are present at discrete sites, for example, branch points, and have not been observed in those inevitably restricted regions examined by electron microscopy. Histochemical techniques coupled with light microscopy have therefore been applied to the problem. Ultraviolet fluorescence microscopy of serial sections from three vessels, covering lengths of 280, 360,~ and 400 ~m, respectively, and stained for catecholamines using the glyoxylic acid procedure (modified from K6nig, 1979, ttistochemistry 61,
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301-305), failed to reveal any axons close to the vessels, including branch points. In every experiment, the histochemical procedure was verified using an internal control from the rat submandibular salivary gland which possesses a rich adrenergic innervation. Previous trials with reserpine to deplete biogenic amines ablated catecholamine fluorescence in this tissue. It is now necessary to test for the presence of cholinergic fibres, and the acetylcholinesterase procedure (modified from EI-Badawi and Schenk, 1967, J. Histochem. Cytochem. 15, 580-588) has been worked up in experiments with rat submandibular and major sublingual glands, our results agreeing with those of Bogart (1971, Amer. J. Anat. 132, 259-266). This technique will now be applied to human breast material. We thank Dr. K. B. Robinson of Dryburn Hospital for the supply of clinical materials, and the British Heart Foundation for supporting this work.
8. Stimulation o f Presynaptic Adrenergic a-Receptors Inhibits Noradrenaline Release in the Rat Uterine Microcirculation. A. Koo, Department of Physiology, Faculty of Medicine, University of Hong Kong, Sassoon Road, Hong Kong. It has been shown that presynaptic adrenergic a-receptors mediate a negative feedback mechanism leading to inhibition of noradrenaline (NA) release during nerve stimulation (for reviews, see Westfall, 1977, Physiol. Rev. 57, 659-728). Recently, we showed the NA release in rat uterine arterioles when hypogastric nerve was stimulated (Koo, 1980, Microvasc. Res. 20, 257-258) and the NA-activated postsynaptic adrenergic a-receptor constrictor responses in these arterioles (Koo and O, 1979, Microvasc. Res. 17, $36). The present communication is intended to show the presence of presynaptic a-receptors which, on stimulation, inhibit NA release. Twenty-three pentobarbital-anaesthetised female rats (100-200 g) at metoestrus were laparotomised. The mesometrium of the uterus was suffused with Krebs solution, transilluminated, and observed with a video microscope. The diameter of an arteriole (30-40 Ixm) was measured from the video monitor by calipers. The hypogastric nerve was stimulated with square waves of 0.5-msec duration for 20 sec at 20 V, with frequencies varying from 0.8 to 25 Hz, while NA was topically applied in concentrations of 0.01 to 10 IxM. The effects of the selective presynaptic a-receptor agonist (clonidine) and antagonist (yohimbine) on nerve stimulation and NA application were studied by adding varying concentrations (1 to 300 txM) of the respective agents into the suffusing Krebs solution. Results confirmed that both hypogastric nerve stimulation and NA topical application produced constrictor responses in the uterine arterioles. The nerve stimulation frequency-response curve was displaced to the right in the presence of clonidine but shifted to the left in the presence of yohimbine, indicating that selective stimulation of the presynaptic c~-receptors inhibited NA release while selective blockade of these receptors facilitated NA release. In contrast, clonidine did not displace the NA concentration-response curve and yohimbine shifted the NA curve to the right, confirming that the postsynaptic a-receptors were not involved in the negative feedback mechanism of presynaptic a-receptor inhibition of NA release. Supported by Astra Pharmaceuticals International.
9. The Rate o f Oxygen Exchange from Erythrocytes to Tissues. J. A. Sins, Department of Biophysics, St. Mary's Hospital Medical School, Paddington, London W2 1PG, London. Though the uptake of oxygen in the lung is considered as being equally limited by the diffusion resistance of the lung membrane, Dm, and the rate of uptake by red blood cells, O, no similar analysis has been made of oxygen supply to tissues. The present analysis is an extension of the numerical method for solving the differential equations involving diffusion and chemical reaction, used by Nicholson and Roughton to interpret oxygen exchange by red blood cells. The rate of egress from red blood cells to tissues was considered as a process of diffusion and chemical dissociation within the internal haemoglobin, followed by diffusion through the cell membrane, plasma and tissue resistance. Allowance was made for the variation of the forward and back reaction velocity constants over the physiological range. Computation shows that the rate of egress would not be sufficient to reach equilibrium within 0.4 sec if diffusion through the erythrocyte haemoglobin or membrane were limiting factors. The tissue diffusion resistance must also be less than that for the lung membrane,
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Din. The calculations imply that the rate of oxygen exchange by red blood cells is only limited by the chemical reactions, and not by diffusion. Physically this arises because of the sigmoid nature of the oxygen dissociation curve. Any significant diffusion resistance would prevent the oxygen pressure within the cell falling below the flattened upper region of the curve, restricting oxygen dissociation.
10. Diameters and Branching Angles of Blood Vessels in the Chick Chorioallantoic Membrane. G. HOOPER, I. MCCARTHY, AND R. FLEMING, Department of Orthopaedic Surgery, Clinical Research Unit, Princess Margaret Rose Orthopaedic Hospital, Edinburgh, Scotland. Murray (1926, J. Gen. Physiol. 9, 835-841) derived theoretical relationships between the diameter of blood vessels and their branching angles at divisions, based on functional considerations. We have measured the diameters and branching angles of vessels (<1 mm diameter) in the chorioallantoic membrane of 10-day-old fertilised eggs. Our results indicate that the relationship between the diameters of parent and daughter vessels at bifurcations is close to the optimal relationship proposed by Murray. However, the branching angles showed a wide scatter about the predicted values.
11. Digital Haemodynamics and Prostaglandin E1 Infusion in Raynaud's Phenomenon. J. E. TOOKE AND M. F. R. MARXIN, University Department of Medicine. The General Infirmary, Great George Street, Leeds LS1 3EX, England. Previous work suggests prostaglandin E~ infusion may be a valuable treatment for peripheral ischaemia. This study was designed to investigate whether prostaglandin E~ infusion in a homogeneous group of patients with Raynaud's phenomenon resulted in (i) subjective benefit, (ii) objective improvement in digital circulation. Subjects comprised six women aged 30 to 74 years all with Raynaud's phenomenon secondary to systemic sclerosis. Vascular measurements were made following acclimatization in a constant-temperature room at 24° before, during, 1 week after, and 6 weeks after a 72-hr intravenous infusion of prostaglandin E~ (maximum infusion rate 10 ng/kg/min). At each study visit nailfold capillary pressure was determined using the Landis microinjection technique. Digital blood flow was determined using an ecg-triggered mercury strain gauge plethysmograph. Finger temperature was measured using adherent thermocouples and radiometry. All subjects had nailfold capillary abnormalities characteristic for systemic sclerosis including reduced capillary numbers, haemorrhages, and bizarre widened forms. Results (means, n = 6)
Arterial limb capillary pressure (cm H20) Capillary pressure gradient (cm H20) Rest flow (ml/100 ml tissue/min) Skin temperature (°)
Pre
During
1 week
6 weeks
30.3
44.1"
40.3
33.1
0
11.7*
2.9
4.0
5.1
9.0
4.3
7.6
28.8
29.8*
29.3
28.9
* Statistically significantly different from preinfusion values, P < 0.05. Four subjects claimed reduction in pain and frequency of attacks of vasospasm. Ischaemic ulcers in two subjects healed. Clinical improvement correlated well with improvement in capillary pressure gradient. This study provides clinical and experimental evidence of improvement in digital nutritional circulation following PGE~ infusion in patients with Raynaud°s phenomenon secondary to systemic sclerosis.
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12. Measurement of Bone and Muscle Perfusion in the Rabbit Using Radioactive Microspheres. I. H. THOMAS, I. HOLLOWAV, ANO D. N. WALDER, University Departments of Surgery and Medical Physics, Royal Victoria Infirmary, Newcastle upon Tyne NEI 4LP, England. The rabbit has often been used as an experimental animal but there is little detailed information available in the literature concerning long bone perfusion. In this study the microsphere method was used to determine the cardiac output, perfusion of the rectus femoris muscle and femur. Ten mature female New Zealand white rabbits were anaesthetised, intubated, and ventilated. Microspheres 15 ~m in diameter labeled with strontium-85 (3M Company) were injected through a catheter inserted directly into the left atrium. Fine bore catheters placed in both brachial arteries were used to measure blood pressure and to obtain "reference organ" blood samples. The animals were then killed and the radioactivity present in the "reference organ," bones, and muscles was measured in a gamma counter designed for bulky specimens. The cardiac output and perfusion of the tissues of interest were then calculated and the following values (mean --- SD) obtained:
Cardiac output Rectus femoris muscle Femoral cortex Femoral marrow
55.8 2.8 3.3 14.1
_ 21.5 ml/min/kg -+ 1.3 ml/min/100 g + 1.7 ml/min/100 g _ 6.5 ml/min/100 g
The regional perfusion of the femoral cortex and marrow was also obtained by dividing the bones into four sections: head and trochanters, upper shaft, lower shaft, and condyles.
13. The Role of Skeletal Blood Flow in Determining the Uptake of99mTc-Methylene Diphosphonate. I. D. McCARTHY AND S. P. E. HUGHES, Department of Orthopaedic Surgery, University of Edinburgh, Edinburgh, Scotland. The relationship between skeletal blood flow and mineral deposition in bone has important consequences for bone physiology, and also for the clinical interpretation of radionuclide bone scans. We have studied the single-passage extraction of 99mTc-methylene diphosphonate in the canine tibia at different flow rates. Extraction decreased with increasing flow through the bone. There was no evidence that the surface area available for exchange increased in response to the increase in perfusion pressure.
14. The Influence of Humoral Factors on Capillary Filtration Coefficient (CFC). P, D. WATSON, Department of Physiology, School of Medicine, University of South Carolina, Columbia, S.C. 29208. In addition to the well-known effects of calcium, magnesium, and protein, Diana et al. have reported a fourth endogenous substance which maintains normal capillary permeability (1981, Microvasc. Res. 21, 241). The present study has verified .this in the cat hindlimb. The isolated limb is perfused, at constant flow from a reservoir-oxygenator system, with an appropriate electrolyte solution containing dialysed bovine serum albumin and a variable quantity of blood. Papaverine created a maximum vasodilation. CFC was calculated from the weight changes following a venous pressure increase of 8 to 10 mm Hg. In four limbs the initial quantity of blood was low, hematocrit 1 to 4%. After measuring CFC, more blood was added to raise the hematocrit to 10 to 14%. In all four, CFC dropped significantly, typically to half the original value. In another five limbs, after measuring CFC with a blood-albumin mixture, perfusing with a blood-free albumin solution always raised the CFC. In two of these cases, plasma was then added, and in both, the CFC returned close to the original value. Other measurements and calculations indicate that viscosity is not a primary effect. Possible causes for the observations are histamine-catecholamine interactions, platelets, and others.
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15. Macrophages in the Circulating Blood (Demonstration). A. S. TODD, Department of Pathology, University of Dundee, P. O. Box 120, Dundee DD1 9SY, Scotland. Smears of the buffy coat are routinely used to examine large numbers of nucleated cells from the circulating blood. Over the last 2 years I have found macrophages in such preparations made from the blood of 31 patients. In 16 cases there was severe infection, in 13 there was peritonitis, and in 11 there was liver disease. The macrophages often contained platelets, red cells, and haemosiderin. They were often foamy but rarely contained lipid. The origin, mechanism of release, and significance of these cells are unknown.