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Abstracts of papers presented at a meeting of the British microcirculation society
MICROVASCULAR
RESEARCH
Abstracts March
20,
253-259
(1980)
of Papers Presented at a Meeting British Microcirculation Society
21 and 22, 1980, St. Mary’s Hospital London, England
Medical
of the School,
Study of Arterioles in Human Breast Biopsy Specimens. C. J. JONES AND J. E. Department of Zoology, University of Durham, Science Laboratories, South Road, Durham DHl 3LE, England.
1. An Ultrastructural KENDALL,
This work forms part of a project to study the structure and organisation of blood vessels in a number of human tissues, both normal and diseased, in an attempt to understand some of the constraints which might operate in the regulation of blood flow. Tissue specimens, obtained from patients with invasive ductal cancer of the breast, were fixed, dehydrated, and embedded in Epon. Arterioles were then located and prepared for electron microscopy. Several vessels were examined in material from four patients (aged 44-63 years) and the following features observed. Endothelial cells, often linked by interdigitations and showing occasional paramembranous densities along the cell junctions, were found to possess a rich and varied cytoplasm. In addition to mitochondriaand vesicles, free and bound ribosomes were observed together with dense nets of microfibrils, the latter possibly contributing to cell structure and affording a means of intracytoplasmic compartmentation. Golgi complexes were also observed. In the endothelial nuclei, the nucleoplasm was surrounded by a characteristic dense band immediately within the nuclear membrane. The smooth muscle cells, rich in myofibrils and micropinocytotic vesicles, often formed discontinuous layers in the smaller vessels, exposing parts of the endothelium. Autonomic fibres were not observed around any of the vessels examined, suggesting that flow was not mediated by nerves running along the vessel length. The possibility that flow might be controlled by innervation at discrete sites is now being investigated. Supported by the British Heart Foundation. 2. The Ultrustructure
of Endocrine
and Exocrine
Capillaries
ill the Puncrras.
J. R.
HENDERSON
AND
M. C. Moss, Department of Physiology, St. George’s Hospital Medical School, London SW17 ORE, England The presence of fenestrae in capillary endothelia is said to be associated with large fluxes of water and/or solutes across the capillary wall. Capillaries in both endocrine and exocrine glands are usually fenestrated. The pancreas is an interesting organ in which to examine these propositions, for it has capillaries supplying the endocrine part of the gland in series with capillaries supplying the exocrine part (Fraser and Henderson, 1979,5. Physiol. 292,4P). Using electron microscopy, we have compared the ultrastructure of the two sorts of capillaries using rat pancreas fixed (by immersion) in glutaraldehyde. The most striking difference between them is the far greater number of fenestrae present in endocrine capillaries: in the 40 capillaries so far examined, there are approximately seven times as many fenestrae in endocrine as in exocrine capillaries. Perhaps the most relevant metabolic difference between the two sorts of tissue is the passage of polypeptide hormones (~7000 daltons) into the lumina of the endocrine capillaries. Is the greater number of fenestrae in endocrine capillaries related to the passage of these polypeptide hormones across their walls’? 253 W262862/80/050253-07$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights uf reproduction in any form reserved. Printed in U.S.A.
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in Capillary Growth in Skeletal Muscle. 0. HUDLICKA,* L. DoDD,t S. D. GRau,t E. M. RENKIN,? W. SCHROEDER,$K. R. TYLER,* AND A. J. A. WRIGHT,* *Department of Physiology, University of Birmingham, Birmingham B15 2TJ, England, tDepartment of Human Physiology, University of California, Davis, California, and $Department of Physiology, J. W. Goethe University, Frankfurt/Main, West Germany.
3. Factors Involved
Indirect electrical stimulation of fast skeletal muscle induces capillary growth and increases activity of oxidative enzymes. In muscles stimulated at slow-nerve frequency (IO Hz) for 8 hriday, capillary growth starts within 4 days, preceding enzyme changes (Brown et al., 1976, P’tigers Arch. 361, 241-250): if the frequency resembles that in nerves to fast muscle (three trains at 40 Hzimin) capillary growth begins later, after increase in oxidative enzyme activity (Hudlicka and Tyler, 1980,J. Physiol. in press). Several factors may be involved in capillary growth. New capillaries always start to grow at the points of maximum curvature of preexisting capillaries (Myrhage and Hudlicka, 1978,Microvusc. Res. 16, 73-90) which suggests that mechanical factors associated with increased blood flow in contracting muscles could be involved. In the present experiments, increased capillary density was indeed found after chronic application of vasodilating drugs. Muscle hypoxia resulting from excessive oxygen demand during prolonged contractions may also be important. Capillary growth starts in the vicinity of fast glycolytic fibres where capillary density is normally low and which are activated throughout the time of stimulation-unlike in exercise. Muscle oxygen tensions (measured using a Pt electrode) are lower during the first few days of stimulation at IO Hz. It is assumed that a combination of relative muscle hypoxia and increased blood flow are factors involved in capillary growth in electrically stimulated muscles.
of the Venules in Skeletal Muscle during Muscle Contraction. J. M. MARSHALL AND H. C. TANDON, Department of Physiology, The Medical School, Birmingham B15 2TJ, England.
4. Responses
Arterioles in skeletal muscle dilate during muscle contraction leading to an increase in blood flow but little is known of the behaviour of venules. We have made direct observations of the microvasculature of spinctrapezius muscles of anaesthetised rats and have found that venules also dilate. Contractions of four to six muscle fibers were induced with pulses of 8-10 V and 0.1 msec duration passed through a monopolar tungsten microelectrode inserted into the muscle. Stimulation for 10 set at 3-8 Hz dilated all arterioles and venules within the region of muscle contraction. Maximum increases in vessel internal diameter were 40% in both smaller arterioles and venules (
5. Digital
Cyclical changes in peripheral blood flow have been previously described during the menstrual cycle, and premenstrual tension may relate to excessive accumulation of fluid prior to the onset of menstruation. As capillary pressure is the most variable determinant of fluid filtration and oedema formation it was decided to study this factor throughout the menstrual cycle. Ten normal healthy wqmen, age 20 to 44 years, mean age 28.8 years, and five women on the combined contraceptive pill, age 20 to 30 years, mean age 25 years, were studied on Days 7, 14,21, and 28 of the menstrual cycle. The day and contraceptive status were unknown to the investigator. Measurements were made in a constant temperature room under standardized conditions. Digital blood flow was determined using mercury strain gauge plethysmography. Capillary pressure was determined using the Landis microinjection
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technique. In normal women there was slight cyclical variation in skin temperature and capillary pressure, with higher values on Days 7 and 2 I, and lower values on Days 14 and 28, but these changes did not achieve statistical significance. In contrast women on the “pill” exhibited higher skin temperatures, digital flows and capillary pressures with an apparent lack of cyclical variation. Values on Day 28 for skin temperature, flow, and capillary pressure were statistically significantly higher than corresponding values in normal women. The results suggest a modulating effect of sex steroids on peripheral blood flow and capillary pressure.
6. The Effects of Temperature on the Transport of Ferritin through Endothelial Cell Vesicles. G. CLOUGH AND C. C. MICHEL, University Laboratory of Physiology, Oxford OX1 3PT, England.
Previously we have demonstrated by electron microscopy that when single frog capillaries are perfused with ferritin solutions for varying periods at 17”, the vesicles of the endothelial cells become progressively labelled with ferritin, reaching steady levels at 40 sec. Some labelling of vesicles at the luminal surface and in the cytoplasm are seen at the earliest times (< I-set perfusion). At all times there is a gradient of labelling across the cell, with the luminal vesicles the most heavily labelled (Clough and Michel, 1979, J. Physiol. 292, 61P). Recently we have carried out similar experiments at 4-7”. Although a gradient of labelling is maintained, the fraction of vesicles labelled is reduced and similar to that seen at i I set, at 17”. It does not increase with increasing perfusion times. We conclude that cooling inhibits movement and fusion of vesicles and that the labelling of cytoplasmic and abluminal vesicles observed at 4-7” is the result of stable communicating vesicular channels across the cell.
7. The Effect qf EDTA on the Endothelial Changes Induced in Postcapillary Venules by Histamine. R. H. A. LIDDELL AND J. G. SIMPSON,Department of Pathology, University of Aberdeen, For-
esterhill, Aberdeen AB9 2ZD, Scotland. Under the influence of the chemical mediators of inflammation, the endothelial cells of postcapillary venules undergo a number of structural changes with dilation and increased permeability of the interendothelial cell junctions. It has been suggested that these arise as a result of active contraction of the endothelial cell: the presence in endothelium of filaments of dimensions and antigenicity similar to the myofilaments of muscle cells supports this view. Endothelial contractility has, however, never been proven. Since a muscle-like contractile mechanism would require calcium, we have tested the effect of calcium removal on histamine-induced endothelial change. Histamine was microinjected into the dissected hamster cheek pouch and its effects studied by electron microscopy. After histamine, the endothelium of postcapillary venules was thicker than normal and the interendothelial junctions were widened and permeable to intravascular carbon. Pretreatment of the cheek pouch with EDTA prevented both the structural changes and the increased permeability. The results suggest that calcium is necessary for the action of histamine on postcapillary venular endothelium and support the theory that endothelial cells are contractile.
of Changes of Permeubility in Single Frog Capillaries. M. E. PHILLIPS AND C. C. MICHEL, University Laboratory of Physiology, Oxford OX1 3PT, England.
8. The Measurement
Using the methods of Michel et al. (1974, Q.J. Exp. Physiol. 59, 283-309), we have measured the filtration coefficient (L,) of single frog mesenteric capillaries perfused with Ringer solutions containing 4% bovine serum albumin before and after the tissue temperature was raised rapidly from 10-20” to 30-35”. The effective osmotic pressure (UT) of the albumin across the capillary wall was also estimated. Following the rise of tissue temperature, L, rose progressively (a sixfold increase being recorded after 20 min) and on fell (reaching 25% of its control value at 20 mitt). Significant changes in L, were seen as early as 90 set after the rise of temperature. Control measurements at 10-20” showed no consistent variation over similar periods of observation. There was a linear relation between the rise in L, and the fall of on. The presence of vinblastine ( 10m5M) and high calcium (IO-* M) in the perfusate decreased the fall in on for a given increase in L,.
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9. The Effect of Magnesium and @starnine on Transmesothelial Protein Flux in the Rat: A Peritoneal Dialysis Study. J. GAMBLE, Department of Physiology, Medical College of St. Bartholomew’s
Hospital, Charterhouse Square, London EClM 6BQ, England. In the isolated perfused rat mesentery, magnesium depletion results in an increased transmesothelial fluid flux and the fluid contains transuded vascular proteins (Gamble et al., 1979,J. Physiol. 2%,8 IP; Microvasc. Res. 18, 283-284). These proteins must traverse both endothelial and mesothelial barriers and the following experiments were performed to study some factors influencing the mesothelial barrier. A sterile cannula was inserted into the peritoneal cavity of sodium pentobarbitoneanesthetized rats and T-1824 injected intravenously to label the plasma proteins. Heparinised Krebs solution, at pH 7.4 and 37” was injected into the peritoneal cavity in IO-ml aliquots, left for I.5 min and then syphoned out over a period of 5 min. Either normal or isomolar magnesium-free Krebs was used, and in either case, after four dialyses, I pg ml-’ histamine was added to subsequent aliquots. Samples of dialysate and arterial blood were centrifuged and the supernatant was subjected to calorimetric, spectrophotometric, and colloid osmotic pressure analysis. Trichloracetic acid precipitation indicated that all plasma and dialysate T-1824 was protein bound. The results showed that neither magnesium depletion nor histamine addition alone caused a significant increase in dialysate protein content. Histamine, however, did cause an increase in protein flux in the absence of magnesium. It is conjectured that magnesium may protect against histamine-mediated increases in protein permeability. IO. The Control
of Fluid Movement across Rabbit Synovium by Intravascular Pressures. A. D. KNIGHT AND J. R. LEVICK, Department of Physiology, St. George’s Hospital Medical School,
London SW17 ORE, England. Synovial fluid is considered to be a modified plasma ultrafiltrate within an enlarged interstitial space. A crucial test of this hypothesis is whether transsynovial flow (Q,) varies with capillary hydrostatic (P,) and oncotic pressure (II,). Transsynovial flow (absorption) of Krebs solution under 18 cm H,O intraarticular pressure was measured by a drop counter connected to an intraarticular cannula (Levick, 1979, J. Physiol. 289, 69-82) from knees of isolated, maximally vasodilated rabbit hindquarters. Hindquarters were perfused with oxygenated human blood (36-39”, pH 7.2-7.4) by a peristaltic pump. Aortic (PA) or inferior caval pressure (PV) was changed in steps every 5-10 min by an arterial Starling resistor or alteration of venous outflow height. Absorption rate Q, declined as a linear function ofPv of mean slope (Sv) -0.21 @min.mm Hg (SE + 0.04, n = 12): and as a function ofP, of slope (S,) - 0. I5 2 0.02 ~l/min.mm Hg at 80-180 mm Hg (n = 12). The ratio S,/S, was pre- to postcapillary resistance ratio, R,/RV, and averaged 1.40 (n = IO). From PA, P,, and RJR,, Pc was calculated. 0. declined as a linear function ofPc at 0.33 @Urnin. mm Hg (SE ? 0.06, II = 10). Perfusate oncotic pressure (measured by a Hansen osmometer) was varied by resuspending bovine erythrocytes in I- I2 g% bovine albumin solution. & increased as HP increased, by 0.31 2 0.06 pl/min. Hg (n = 5). The near equality of do,/dPc and d&/d&, indicated a reflection coefficient of -I for bovine albumin at synovial endothelium. The results demonstrate regulation of synovial fluid flow by vascular pressures and support a recent model of transsynovial flow (Levick, 1979,J. Physiol. 290, 34-35P). Il. Low Permeability
to Macromolecules of the Fenestrated Capillaries in the Cat Mandibular Salivary Gland. A. Koo, L. H. SMAJE, K. A. DZIELGIELEWSKA,* P. H. SPENCER,AND W. P. PENN, Department of Physiology, Charing Cross Hospital Medical School, Fulham Palace Road,
London W6 8RF, and *Department of Physiology, University College London, Gower Street, London WCIE 6BT, England. Recently Mann et al. (1979, J. Physiol. 297, 335-354) showed that permeability-surface area products (PS) in the cat mandibular salivary gland for “‘Cr-EDTA, cyano[57Co]-cobalamin and lz51labelled insulin were 100-200 times those in skeletal muscle but there was no evidence for an equivalent increase in albumin permeability. The present experiments were designed to specifically measure macromolecular permeability by collecting lymph from the gland and obtaining PS from the lymph:plasma ratio and lymph flow. PS = LRI(I-R) where L is lymph flow and R is lymph:plasma ratio. In anesthetized cats the gland was exposed and one of the three or four draining lymphatics identified and cannulated, and lymph was collected in tared plastic vials. Albumin and IgG were estimated using an immunodiffusion technique. In seven cats investigated the maximum lymph flow
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was 5.6 pl/min/g assuming four lymphatics, and lymph:plasma ratios were 0.25 t 0.014 for albumin and 0.19 2 0.012 for IgG. These measurements give maximumPS values of 1.8 X 10m3mUmin/g for albumin and 1.3 x IO-” mliminlg for IgG. Such values are within the range quoted for intestine and other nonfenestrated tissues (see Renkin, 1977,Circ. Res. 41,735-743) and are not consistent with a uniform pore size of 12 nm as determined by Mann et (I/. (lot. cit.) on the basis of restricted pore theory. It is also difficult to fit the data to a simple two-pore diffusion model and alternatives are being explored. 12. The Acid-Base Balance of Intersritiul Fluid. .I. A. SIRS, Department of Biophysics, St Mary‘s Hospital Medical School, University of London, London W2 IPG, England. The acid-base balance of blood is assessed from the chemical reaction of carbon dioxide with water CO, + HZ0 = H&O, G= HCO, + H+ + BII HB Using the Henderson-Hasselbalch
(1)
equilibrium relationship
where [HCO;] is in millimolar, and PCO, in millimeters of Hg. The speed of the chemical reaction (1) depends on the strength of the buffer, the greater the concentration of buffer protein (HB) the slower is the reaction rate. Due to the presence of carbonic anhydrase the reaction is 50% complete, within the erythrocyte, in a few milliseconds, and the plasma bicarbonate level correspondingly changes via the Cl-IHCO; exchange. In the plasma, the buffer/power of the plasma proteins slows the reaction, and pH equilibrium is not attained during circulation. Equation (2) does not apply to blood it7 tivo. During capillary exchange plasma proteins are retained in the capillary, leaving interstitial fluid with a low buffer capacity. The reaction of arterial plasma bicarbonate, HCO;, with tissue CO, is then rapid, within a fraction of a second, and the change of bicarbonate involved is negligible. The pH of interstitial fluid can thus be obtained by inserting LHCO;], and tissue (venous) PCO, into Eq. (2). Lowering [HCO;],, by hyperventilation or rapid ascent to altitude, causes tissue acidosis, not alkalosis as commonly stated. Preventing the bicarbonate fall, by partially inhibiting carbonic anhydrase, will reduce the tissue acidosis. 13. Relectse of Norcrdrenoline in the Arterioles in the Mrsometrium of the Rut Uterus. A. KOO, Department of Physiology, Faculty of Medicine, University of Hong Kong, Sassoon Road, Hong Kong. We have shown in the rat uterus that arterioles in the microcirculation of the mesometrium possess a-adrenoceptors. Topical application of noradrenaline leads to constriction of the arterioles (Koo and 0, 1979, Microvasc. Res. 17, S36) and stimulation of the hypogastric (sympathetic) nerve produces vasoconstriction in cocainized preparations (Koo and 0. 1979,“Society for Study of Fertility,” Glasgow Meeting Abstr., p.27). The present communication shows the release of noradrenaline by tyramine in cocainized and reserpinized preparations. A total of 30 pentobarbital-anesthetized female rats (120 g) at early proestrus were laparotomized. The mesometrium of the uterus was suffused with Krebs solution, transilluminated, and observed with a video microscope system. The diameter (60-120 pm) of an arteriole was measured from the video monitor by a pair of calipers. Neuronal reuptake of noradrenaline from the nerve ending was blocked by topically applied cocaine (IO-“- 10e2moliliter) for 30 min to the mesometrial preparation, and depletion of noradrenaline stores was produced using reserpine (IO-” moliliter) for 90 min. Results show that noradrenaline ( 1O-9-10m2moliliter) produces concentration-dependent vasoconstriction in normal, cocainized and reserpinized preparations. However. both stimulation (I - 16 Hz) of hypogastric nerve and topical application (IO-l”- IO-“mol/liter) of tyramine produce vasoconstriction only in cocainized preparations. These findings suggest that norad-
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renaline can be relased from the sympathetic nerve endings but probably the noradrenaline cannot reach the vascular neuroeffector site in the uterine arterioles because of the neuronal reuptake mechanism. 14. An EM Histochemical Sublingual
Glands.
Study of Perivascular Nerves in the Rat Submandibular and Major C. J. JONES, Department of Zoology, University of Durham, Science
Laboratories, South Road, Durham DHl 3LE, England. Recent excitement over the possibility of novel “peptidergic” or “purinergic” components in the autonomic nervous system has prompted an ultrastructural study of perivascular innervation in the rat submandibular and major sublingual glands, where atropine-resistant parasympathetic vasodilation has been reported. Glands were removed from freshly killed rats and subjected to the modified chromaflin procedure to distinguish autonomic subpopulations in the electron microscope (Jones, 1979,Neurosci. Lett. 13, 19-23). Arterioles were found to be richly innervated and venules rarely so. Axon profiles containing a predominance of dense-cored vesicles and thought to be monoaminergic were observed around arterioles in both glands and were not seen following in vivo treatment with reserpine to deplete biogenic amines or with 6-hydroxydopamine to chemically sympathectomise. Profiles containing a predominance of unstained vesicles and thought to be cholinergic were also observed, frequently around arterioles in the submandibular gland, but rarely in the major sublingual gland where, instead, nonadrenergic noncholinergic axon types containing large grey-cored vesicles were occasionally observed. This finding harmonises with the report of fibres containing vasoactive intestinal polypeptide (VIP)-like immunoreactivity in the same location (Wharton et al., 1979,Life Sci. 25, 273-280) but it is too early to say whether the two populations are the same. It seems unlikely, however, that these structures could explain atropine resistance because of their apparent absence from blood vessels in the submandibular gland. The support of the Addison Wheeler Fellowship, University of Durham, is gratefully acknowledged. 15. Bulk Flow through the Sclera. D. F. London WClH 9QS, England.
COLE AND
K.
OHARA,
Institute of Ophthalmology, Judd Street,
These experiments were undertaken to assess the ability of the scleral coat of the eye to permit the bulk flow of fluid and solutes. The hydraulic conductivity of isolated pieces of sclera was measured as described previously (Cole, 1978, Ophthal. Res. 10, 279-282) and the penetration of various sizes of fluorescein isothiocyanate-labelled dextran (FITC-dextran) estimated by maintaining a known concentration on the inner side of the tissue and calculating the steady-state concentration in the effluent fluid from the outer surface. In separate experiments using intact rabbits, radioiodine-labelled albumin was introduced either into the anterior chamber of the eye or, via the cerebrospinal fluid and optic nerve sheath, at the posterior pole. Penetration of the label into the sclera was investigated by comparing the radioactivities of the anterior and posterior regions. The hydraulic conductivity of the sclera in vitro was 7.61 ? 0.97 nllminimm Hg/cm2 (mean f SE) and there was significant penetration of FITC-dextrans, the steady-state concentration ratios being 58.6 2 5.5% for FITC-3 and 27.5 2 7.4% for FITC-150. In the second series of experiments the direction of the concentration gradient for labelled albumin (i.e., whether anteroposterior or posteroanterior) depended on the site at which it was introduced. Bulk flow of water and large molecules in the sclera can occur either radially or meridionally and may contribute to the normal drainage of the intraocular fluids. K. Ohara is supported by a Wellcome Research Fellowship (Japan). 16. Mechanical versus Biochemical Factors in Angiogenesis. T. J. RYAN Department of Dermatology, The Slade Hospital, Oxford, England.
AND
A.
T. STOCKLEY,
At present angiogenesis is considered to be due to a chemical angiogenic factor. However, for almost 100 years the role of mechanical factors has been discussed. These include intravascular pulsation, decreased tissue pressure, or contact with some scaffold material such as fibrin or collagen. The role of mechanical factors in angiogenesis in the hamster cheek pouch and on the chorioallantoic membrane will be discussed. This is based on past studies of epidermal grafts and homogenates in the hamster cheek pouch (Nishioka and Ryan, 1972,J. Invest. Dermatol. 58,33) and current studies using the chorioallantoic membrane. Graded stresses using various “shapes” of contact with the membrane
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can induce angiogenesis of almost any pattern. A glass ring causes radial angiogenesis around it but none in the centre. “Triangular” injuries to the membrane result in “bunching” of the membrane and radial patterns of angiogenesis. Two such “bunches” placed adjacent to each other cause a papillary pattern of angiogenesis in the intervening tissue. It is not easy to rule out a biochemical stimulus from some intermediary such as distorted epithelium. The similarity of this problem to the problem of angiogenesis in the skin or cornea will be emphasized. 17. A Comparison of the Binding to Erythrocytes of Fibrinogen and Its Plasmin Degradation Products. M. W. RAMPLING AND D. J. R. HENDERSON, Biophysics Department, St Mary’s Hospital
Medical School, London W2 IPG, England A compelling body of data is now available indicating that fibrinogen bound to the erythrocyte membrane can have a significant effect on haemorheology. A study has therefore been undertaken of the factors which affect this binding and, in an attempt to elucidate the binding sites on the molecule involved, fibrinogen degradation products (FDP) produced by plasmin digestion have also been investigated. In all cases the finding was estimated from the partition of iz5I-labelled material between the red cells and suspending phase. It was found that fibrinogen and all the major FDPs bound significantly to erythrocytes, but that the intermediate asymmetric fragment Y did so maximally. The factors affecting the binding were generally similar for all the molecular species investigated. Thus it increased with both decreasing pH (from 8 to 5.4) and with decreasing ionic strength (from 0.2 to 0.1 M) but was independent of temperature (from 4 to 37”) and of calcium ion concentration. Studies using the centrifuge technique have shown that red cell flexibility is influenced by fibrinogen and the large FDPs X and Y, but not by the small late fragments, D and E. Since fibrinogen and fragment X consist essentially of one E and two D fragments covalently linked, while Y consists of one D and one E, the centrifuge results would suggest that at least two widely spaced binding sites are essential if the bound molecules are to affect erythrocyte flexibility. This study was supported by a grant from the Wellcome Trust. and Plasma Fibrinogen in Diabetes. G. D. 0. LOWE, M. SINGARAVELLOO, J. L. S. PARKER, W. G. MANDERSON, C. D. FORBES AND C. R. M. PRENTICE, Department of Medicine and Diabetic Unit, Royal Infirmary, Glasgow G4 OSF, Scotland
18. Platelet Aggregates
Increased plasma fibrinogen levels have been described in diabetics and are associated with vascular complications. Platelet abnormalities are also found in diabetes, and recently increased “circulating” platelet aggregates (CPA) have been detected by formalin fixation in freshly drawn blood. As we have previously found a relationship between CPA and plasma fibrinogen levels (Lowe et al., 1979, Thromb. Res. 14, 377-386), we compared CPA and plasma fibrinogen in 22 diabetics with vascular complications (at least two each of atherosclerosis, peripheral neuropathy, retinopathy, or proteinuria), 20 diabetics without complications, and 42 nondiabetics, matched for age, sex, and smoking habit.
Age (years) Fibrinogen (g/l) CPA (% total) Platelets (x lOgil)
Nondiabetics
Diabetics-no complications
Diabeticscomplications
52 (4)” 2.7 (0.1) 13 (2) 260 (15)
47 (4) 3.2 (0.2) 14 (3) 231 (10)
57 (2) 3.4 (0.2) 25 (3) 254 (14)
c( Mean (+ SEM). Fibrinogen was increased in both groups of diabetics compared with matched controls (P < 0.05). CPA were increased only in diabetics with complications (P < 0.01). There were no significant correlations of CPA with fibrinogen level, platelet count, age, or plasma glucose. The increased CPA may be due to other plasma factors, intrinsic platelet abnormality, or vasculopathy.
RESEARCH
Abstracts March
20,
253-259
(1980)
of Papers Presented at a Meeting British Microcirculation Society
21 and 22, 1980, St. Mary’s Hospital London, England
Medical
of the School,
Study of Arterioles in Human Breast Biopsy Specimens. C. J. JONES AND J. E. Department of Zoology, University of Durham, Science Laboratories, South Road, Durham DHl 3LE, England.
1. An Ultrastructural KENDALL,
This work forms part of a project to study the structure and organisation of blood vessels in a number of human tissues, both normal and diseased, in an attempt to understand some of the constraints which might operate in the regulation of blood flow. Tissue specimens, obtained from patients with invasive ductal cancer of the breast, were fixed, dehydrated, and embedded in Epon. Arterioles were then located and prepared for electron microscopy. Several vessels were examined in material from four patients (aged 44-63 years) and the following features observed. Endothelial cells, often linked by interdigitations and showing occasional paramembranous densities along the cell junctions, were found to possess a rich and varied cytoplasm. In addition to mitochondriaand vesicles, free and bound ribosomes were observed together with dense nets of microfibrils, the latter possibly contributing to cell structure and affording a means of intracytoplasmic compartmentation. Golgi complexes were also observed. In the endothelial nuclei, the nucleoplasm was surrounded by a characteristic dense band immediately within the nuclear membrane. The smooth muscle cells, rich in myofibrils and micropinocytotic vesicles, often formed discontinuous layers in the smaller vessels, exposing parts of the endothelium. Autonomic fibres were not observed around any of the vessels examined, suggesting that flow was not mediated by nerves running along the vessel length. The possibility that flow might be controlled by innervation at discrete sites is now being investigated. Supported by the British Heart Foundation. 2. The Ultrustructure
of Endocrine
and Exocrine
Capillaries
ill the Puncrras.
J. R.
HENDERSON
AND
M. C. Moss, Department of Physiology, St. George’s Hospital Medical School, London SW17 ORE, England The presence of fenestrae in capillary endothelia is said to be associated with large fluxes of water and/or solutes across the capillary wall. Capillaries in both endocrine and exocrine glands are usually fenestrated. The pancreas is an interesting organ in which to examine these propositions, for it has capillaries supplying the endocrine part of the gland in series with capillaries supplying the exocrine part (Fraser and Henderson, 1979,5. Physiol. 292,4P). Using electron microscopy, we have compared the ultrastructure of the two sorts of capillaries using rat pancreas fixed (by immersion) in glutaraldehyde. The most striking difference between them is the far greater number of fenestrae present in endocrine capillaries: in the 40 capillaries so far examined, there are approximately seven times as many fenestrae in endocrine as in exocrine capillaries. Perhaps the most relevant metabolic difference between the two sorts of tissue is the passage of polypeptide hormones (~7000 daltons) into the lumina of the endocrine capillaries. Is the greater number of fenestrae in endocrine capillaries related to the passage of these polypeptide hormones across their walls’? 253 W262862/80/050253-07$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights uf reproduction in any form reserved. Printed in U.S.A.
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in Capillary Growth in Skeletal Muscle. 0. HUDLICKA,* L. DoDD,t S. D. GRau,t E. M. RENKIN,? W. SCHROEDER,$K. R. TYLER,* AND A. J. A. WRIGHT,* *Department of Physiology, University of Birmingham, Birmingham B15 2TJ, England, tDepartment of Human Physiology, University of California, Davis, California, and $Department of Physiology, J. W. Goethe University, Frankfurt/Main, West Germany.
3. Factors Involved
Indirect electrical stimulation of fast skeletal muscle induces capillary growth and increases activity of oxidative enzymes. In muscles stimulated at slow-nerve frequency (IO Hz) for 8 hriday, capillary growth starts within 4 days, preceding enzyme changes (Brown et al., 1976, P’tigers Arch. 361, 241-250): if the frequency resembles that in nerves to fast muscle (three trains at 40 Hzimin) capillary growth begins later, after increase in oxidative enzyme activity (Hudlicka and Tyler, 1980,J. Physiol. in press). Several factors may be involved in capillary growth. New capillaries always start to grow at the points of maximum curvature of preexisting capillaries (Myrhage and Hudlicka, 1978,Microvusc. Res. 16, 73-90) which suggests that mechanical factors associated with increased blood flow in contracting muscles could be involved. In the present experiments, increased capillary density was indeed found after chronic application of vasodilating drugs. Muscle hypoxia resulting from excessive oxygen demand during prolonged contractions may also be important. Capillary growth starts in the vicinity of fast glycolytic fibres where capillary density is normally low and which are activated throughout the time of stimulation-unlike in exercise. Muscle oxygen tensions (measured using a Pt electrode) are lower during the first few days of stimulation at IO Hz. It is assumed that a combination of relative muscle hypoxia and increased blood flow are factors involved in capillary growth in electrically stimulated muscles.
of the Venules in Skeletal Muscle during Muscle Contraction. J. M. MARSHALL AND H. C. TANDON, Department of Physiology, The Medical School, Birmingham B15 2TJ, England.
4. Responses
Arterioles in skeletal muscle dilate during muscle contraction leading to an increase in blood flow but little is known of the behaviour of venules. We have made direct observations of the microvasculature of spinctrapezius muscles of anaesthetised rats and have found that venules also dilate. Contractions of four to six muscle fibers were induced with pulses of 8-10 V and 0.1 msec duration passed through a monopolar tungsten microelectrode inserted into the muscle. Stimulation for 10 set at 3-8 Hz dilated all arterioles and venules within the region of muscle contraction. Maximum increases in vessel internal diameter were 40% in both smaller arterioles and venules (
5. Digital
Cyclical changes in peripheral blood flow have been previously described during the menstrual cycle, and premenstrual tension may relate to excessive accumulation of fluid prior to the onset of menstruation. As capillary pressure is the most variable determinant of fluid filtration and oedema formation it was decided to study this factor throughout the menstrual cycle. Ten normal healthy wqmen, age 20 to 44 years, mean age 28.8 years, and five women on the combined contraceptive pill, age 20 to 30 years, mean age 25 years, were studied on Days 7, 14,21, and 28 of the menstrual cycle. The day and contraceptive status were unknown to the investigator. Measurements were made in a constant temperature room under standardized conditions. Digital blood flow was determined using mercury strain gauge plethysmography. Capillary pressure was determined using the Landis microinjection
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technique. In normal women there was slight cyclical variation in skin temperature and capillary pressure, with higher values on Days 7 and 2 I, and lower values on Days 14 and 28, but these changes did not achieve statistical significance. In contrast women on the “pill” exhibited higher skin temperatures, digital flows and capillary pressures with an apparent lack of cyclical variation. Values on Day 28 for skin temperature, flow, and capillary pressure were statistically significantly higher than corresponding values in normal women. The results suggest a modulating effect of sex steroids on peripheral blood flow and capillary pressure.
6. The Effects of Temperature on the Transport of Ferritin through Endothelial Cell Vesicles. G. CLOUGH AND C. C. MICHEL, University Laboratory of Physiology, Oxford OX1 3PT, England.
Previously we have demonstrated by electron microscopy that when single frog capillaries are perfused with ferritin solutions for varying periods at 17”, the vesicles of the endothelial cells become progressively labelled with ferritin, reaching steady levels at 40 sec. Some labelling of vesicles at the luminal surface and in the cytoplasm are seen at the earliest times (< I-set perfusion). At all times there is a gradient of labelling across the cell, with the luminal vesicles the most heavily labelled (Clough and Michel, 1979, J. Physiol. 292, 61P). Recently we have carried out similar experiments at 4-7”. Although a gradient of labelling is maintained, the fraction of vesicles labelled is reduced and similar to that seen at i I set, at 17”. It does not increase with increasing perfusion times. We conclude that cooling inhibits movement and fusion of vesicles and that the labelling of cytoplasmic and abluminal vesicles observed at 4-7” is the result of stable communicating vesicular channels across the cell.
7. The Effect qf EDTA on the Endothelial Changes Induced in Postcapillary Venules by Histamine. R. H. A. LIDDELL AND J. G. SIMPSON,Department of Pathology, University of Aberdeen, For-
esterhill, Aberdeen AB9 2ZD, Scotland. Under the influence of the chemical mediators of inflammation, the endothelial cells of postcapillary venules undergo a number of structural changes with dilation and increased permeability of the interendothelial cell junctions. It has been suggested that these arise as a result of active contraction of the endothelial cell: the presence in endothelium of filaments of dimensions and antigenicity similar to the myofilaments of muscle cells supports this view. Endothelial contractility has, however, never been proven. Since a muscle-like contractile mechanism would require calcium, we have tested the effect of calcium removal on histamine-induced endothelial change. Histamine was microinjected into the dissected hamster cheek pouch and its effects studied by electron microscopy. After histamine, the endothelium of postcapillary venules was thicker than normal and the interendothelial junctions were widened and permeable to intravascular carbon. Pretreatment of the cheek pouch with EDTA prevented both the structural changes and the increased permeability. The results suggest that calcium is necessary for the action of histamine on postcapillary venular endothelium and support the theory that endothelial cells are contractile.
of Changes of Permeubility in Single Frog Capillaries. M. E. PHILLIPS AND C. C. MICHEL, University Laboratory of Physiology, Oxford OX1 3PT, England.
8. The Measurement
Using the methods of Michel et al. (1974, Q.J. Exp. Physiol. 59, 283-309), we have measured the filtration coefficient (L,) of single frog mesenteric capillaries perfused with Ringer solutions containing 4% bovine serum albumin before and after the tissue temperature was raised rapidly from 10-20” to 30-35”. The effective osmotic pressure (UT) of the albumin across the capillary wall was also estimated. Following the rise of tissue temperature, L, rose progressively (a sixfold increase being recorded after 20 min) and on fell (reaching 25% of its control value at 20 mitt). Significant changes in L, were seen as early as 90 set after the rise of temperature. Control measurements at 10-20” showed no consistent variation over similar periods of observation. There was a linear relation between the rise in L, and the fall of on. The presence of vinblastine ( 10m5M) and high calcium (IO-* M) in the perfusate decreased the fall in on for a given increase in L,.
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9. The Effect of Magnesium and @starnine on Transmesothelial Protein Flux in the Rat: A Peritoneal Dialysis Study. J. GAMBLE, Department of Physiology, Medical College of St. Bartholomew’s
Hospital, Charterhouse Square, London EClM 6BQ, England. In the isolated perfused rat mesentery, magnesium depletion results in an increased transmesothelial fluid flux and the fluid contains transuded vascular proteins (Gamble et al., 1979,J. Physiol. 2%,8 IP; Microvasc. Res. 18, 283-284). These proteins must traverse both endothelial and mesothelial barriers and the following experiments were performed to study some factors influencing the mesothelial barrier. A sterile cannula was inserted into the peritoneal cavity of sodium pentobarbitoneanesthetized rats and T-1824 injected intravenously to label the plasma proteins. Heparinised Krebs solution, at pH 7.4 and 37” was injected into the peritoneal cavity in IO-ml aliquots, left for I.5 min and then syphoned out over a period of 5 min. Either normal or isomolar magnesium-free Krebs was used, and in either case, after four dialyses, I pg ml-’ histamine was added to subsequent aliquots. Samples of dialysate and arterial blood were centrifuged and the supernatant was subjected to calorimetric, spectrophotometric, and colloid osmotic pressure analysis. Trichloracetic acid precipitation indicated that all plasma and dialysate T-1824 was protein bound. The results showed that neither magnesium depletion nor histamine addition alone caused a significant increase in dialysate protein content. Histamine, however, did cause an increase in protein flux in the absence of magnesium. It is conjectured that magnesium may protect against histamine-mediated increases in protein permeability. IO. The Control
of Fluid Movement across Rabbit Synovium by Intravascular Pressures. A. D. KNIGHT AND J. R. LEVICK, Department of Physiology, St. George’s Hospital Medical School,
London SW17 ORE, England. Synovial fluid is considered to be a modified plasma ultrafiltrate within an enlarged interstitial space. A crucial test of this hypothesis is whether transsynovial flow (Q,) varies with capillary hydrostatic (P,) and oncotic pressure (II,). Transsynovial flow (absorption) of Krebs solution under 18 cm H,O intraarticular pressure was measured by a drop counter connected to an intraarticular cannula (Levick, 1979, J. Physiol. 289, 69-82) from knees of isolated, maximally vasodilated rabbit hindquarters. Hindquarters were perfused with oxygenated human blood (36-39”, pH 7.2-7.4) by a peristaltic pump. Aortic (PA) or inferior caval pressure (PV) was changed in steps every 5-10 min by an arterial Starling resistor or alteration of venous outflow height. Absorption rate Q, declined as a linear function ofPv of mean slope (Sv) -0.21 @min.mm Hg (SE + 0.04, n = 12): and as a function ofP, of slope (S,) - 0. I5 2 0.02 ~l/min.mm Hg at 80-180 mm Hg (n = 12). The ratio S,/S, was pre- to postcapillary resistance ratio, R,/RV, and averaged 1.40 (n = IO). From PA, P,, and RJR,, Pc was calculated. 0. declined as a linear function ofPc at 0.33 @Urnin. mm Hg (SE ? 0.06, II = 10). Perfusate oncotic pressure (measured by a Hansen osmometer) was varied by resuspending bovine erythrocytes in I- I2 g% bovine albumin solution. & increased as HP increased, by 0.31 2 0.06 pl/min. Hg (n = 5). The near equality of do,/dPc and d&/d&, indicated a reflection coefficient of -I for bovine albumin at synovial endothelium. The results demonstrate regulation of synovial fluid flow by vascular pressures and support a recent model of transsynovial flow (Levick, 1979,J. Physiol. 290, 34-35P). Il. Low Permeability
to Macromolecules of the Fenestrated Capillaries in the Cat Mandibular Salivary Gland. A. Koo, L. H. SMAJE, K. A. DZIELGIELEWSKA,* P. H. SPENCER,AND W. P. PENN, Department of Physiology, Charing Cross Hospital Medical School, Fulham Palace Road,
London W6 8RF, and *Department of Physiology, University College London, Gower Street, London WCIE 6BT, England. Recently Mann et al. (1979, J. Physiol. 297, 335-354) showed that permeability-surface area products (PS) in the cat mandibular salivary gland for “‘Cr-EDTA, cyano[57Co]-cobalamin and lz51labelled insulin were 100-200 times those in skeletal muscle but there was no evidence for an equivalent increase in albumin permeability. The present experiments were designed to specifically measure macromolecular permeability by collecting lymph from the gland and obtaining PS from the lymph:plasma ratio and lymph flow. PS = LRI(I-R) where L is lymph flow and R is lymph:plasma ratio. In anesthetized cats the gland was exposed and one of the three or four draining lymphatics identified and cannulated, and lymph was collected in tared plastic vials. Albumin and IgG were estimated using an immunodiffusion technique. In seven cats investigated the maximum lymph flow
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was 5.6 pl/min/g assuming four lymphatics, and lymph:plasma ratios were 0.25 t 0.014 for albumin and 0.19 2 0.012 for IgG. These measurements give maximumPS values of 1.8 X 10m3mUmin/g for albumin and 1.3 x IO-” mliminlg for IgG. Such values are within the range quoted for intestine and other nonfenestrated tissues (see Renkin, 1977,Circ. Res. 41,735-743) and are not consistent with a uniform pore size of 12 nm as determined by Mann et (I/. (lot. cit.) on the basis of restricted pore theory. It is also difficult to fit the data to a simple two-pore diffusion model and alternatives are being explored. 12. The Acid-Base Balance of Intersritiul Fluid. .I. A. SIRS, Department of Biophysics, St Mary‘s Hospital Medical School, University of London, London W2 IPG, England. The acid-base balance of blood is assessed from the chemical reaction of carbon dioxide with water CO, + HZ0 = H&O, G= HCO, + H+ + BII HB Using the Henderson-Hasselbalch
(1)
equilibrium relationship
where [HCO;] is in millimolar, and PCO, in millimeters of Hg. The speed of the chemical reaction (1) depends on the strength of the buffer, the greater the concentration of buffer protein (HB) the slower is the reaction rate. Due to the presence of carbonic anhydrase the reaction is 50% complete, within the erythrocyte, in a few milliseconds, and the plasma bicarbonate level correspondingly changes via the Cl-IHCO; exchange. In the plasma, the buffer/power of the plasma proteins slows the reaction, and pH equilibrium is not attained during circulation. Equation (2) does not apply to blood it7 tivo. During capillary exchange plasma proteins are retained in the capillary, leaving interstitial fluid with a low buffer capacity. The reaction of arterial plasma bicarbonate, HCO;, with tissue CO, is then rapid, within a fraction of a second, and the change of bicarbonate involved is negligible. The pH of interstitial fluid can thus be obtained by inserting LHCO;], and tissue (venous) PCO, into Eq. (2). Lowering [HCO;],, by hyperventilation or rapid ascent to altitude, causes tissue acidosis, not alkalosis as commonly stated. Preventing the bicarbonate fall, by partially inhibiting carbonic anhydrase, will reduce the tissue acidosis. 13. Relectse of Norcrdrenoline in the Arterioles in the Mrsometrium of the Rut Uterus. A. KOO, Department of Physiology, Faculty of Medicine, University of Hong Kong, Sassoon Road, Hong Kong. We have shown in the rat uterus that arterioles in the microcirculation of the mesometrium possess a-adrenoceptors. Topical application of noradrenaline leads to constriction of the arterioles (Koo and 0, 1979, Microvasc. Res. 17, S36) and stimulation of the hypogastric (sympathetic) nerve produces vasoconstriction in cocainized preparations (Koo and 0. 1979,“Society for Study of Fertility,” Glasgow Meeting Abstr., p.27). The present communication shows the release of noradrenaline by tyramine in cocainized and reserpinized preparations. A total of 30 pentobarbital-anesthetized female rats (120 g) at early proestrus were laparotomized. The mesometrium of the uterus was suffused with Krebs solution, transilluminated, and observed with a video microscope system. The diameter (60-120 pm) of an arteriole was measured from the video monitor by a pair of calipers. Neuronal reuptake of noradrenaline from the nerve ending was blocked by topically applied cocaine (IO-“- 10e2moliliter) for 30 min to the mesometrial preparation, and depletion of noradrenaline stores was produced using reserpine (IO-” moliliter) for 90 min. Results show that noradrenaline ( 1O-9-10m2moliliter) produces concentration-dependent vasoconstriction in normal, cocainized and reserpinized preparations. However. both stimulation (I - 16 Hz) of hypogastric nerve and topical application (IO-l”- IO-“mol/liter) of tyramine produce vasoconstriction only in cocainized preparations. These findings suggest that norad-
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renaline can be relased from the sympathetic nerve endings but probably the noradrenaline cannot reach the vascular neuroeffector site in the uterine arterioles because of the neuronal reuptake mechanism. 14. An EM Histochemical Sublingual
Glands.
Study of Perivascular Nerves in the Rat Submandibular and Major C. J. JONES, Department of Zoology, University of Durham, Science
Laboratories, South Road, Durham DHl 3LE, England. Recent excitement over the possibility of novel “peptidergic” or “purinergic” components in the autonomic nervous system has prompted an ultrastructural study of perivascular innervation in the rat submandibular and major sublingual glands, where atropine-resistant parasympathetic vasodilation has been reported. Glands were removed from freshly killed rats and subjected to the modified chromaflin procedure to distinguish autonomic subpopulations in the electron microscope (Jones, 1979,Neurosci. Lett. 13, 19-23). Arterioles were found to be richly innervated and venules rarely so. Axon profiles containing a predominance of dense-cored vesicles and thought to be monoaminergic were observed around arterioles in both glands and were not seen following in vivo treatment with reserpine to deplete biogenic amines or with 6-hydroxydopamine to chemically sympathectomise. Profiles containing a predominance of unstained vesicles and thought to be cholinergic were also observed, frequently around arterioles in the submandibular gland, but rarely in the major sublingual gland where, instead, nonadrenergic noncholinergic axon types containing large grey-cored vesicles were occasionally observed. This finding harmonises with the report of fibres containing vasoactive intestinal polypeptide (VIP)-like immunoreactivity in the same location (Wharton et al., 1979,Life Sci. 25, 273-280) but it is too early to say whether the two populations are the same. It seems unlikely, however, that these structures could explain atropine resistance because of their apparent absence from blood vessels in the submandibular gland. The support of the Addison Wheeler Fellowship, University of Durham, is gratefully acknowledged. 15. Bulk Flow through the Sclera. D. F. London WClH 9QS, England.
COLE AND
K.
OHARA,
Institute of Ophthalmology, Judd Street,
These experiments were undertaken to assess the ability of the scleral coat of the eye to permit the bulk flow of fluid and solutes. The hydraulic conductivity of isolated pieces of sclera was measured as described previously (Cole, 1978, Ophthal. Res. 10, 279-282) and the penetration of various sizes of fluorescein isothiocyanate-labelled dextran (FITC-dextran) estimated by maintaining a known concentration on the inner side of the tissue and calculating the steady-state concentration in the effluent fluid from the outer surface. In separate experiments using intact rabbits, radioiodine-labelled albumin was introduced either into the anterior chamber of the eye or, via the cerebrospinal fluid and optic nerve sheath, at the posterior pole. Penetration of the label into the sclera was investigated by comparing the radioactivities of the anterior and posterior regions. The hydraulic conductivity of the sclera in vitro was 7.61 ? 0.97 nllminimm Hg/cm2 (mean f SE) and there was significant penetration of FITC-dextrans, the steady-state concentration ratios being 58.6 2 5.5% for FITC-3 and 27.5 2 7.4% for FITC-150. In the second series of experiments the direction of the concentration gradient for labelled albumin (i.e., whether anteroposterior or posteroanterior) depended on the site at which it was introduced. Bulk flow of water and large molecules in the sclera can occur either radially or meridionally and may contribute to the normal drainage of the intraocular fluids. K. Ohara is supported by a Wellcome Research Fellowship (Japan). 16. Mechanical versus Biochemical Factors in Angiogenesis. T. J. RYAN Department of Dermatology, The Slade Hospital, Oxford, England.
AND
A.
T. STOCKLEY,
At present angiogenesis is considered to be due to a chemical angiogenic factor. However, for almost 100 years the role of mechanical factors has been discussed. These include intravascular pulsation, decreased tissue pressure, or contact with some scaffold material such as fibrin or collagen. The role of mechanical factors in angiogenesis in the hamster cheek pouch and on the chorioallantoic membrane will be discussed. This is based on past studies of epidermal grafts and homogenates in the hamster cheek pouch (Nishioka and Ryan, 1972,J. Invest. Dermatol. 58,33) and current studies using the chorioallantoic membrane. Graded stresses using various “shapes” of contact with the membrane
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can induce angiogenesis of almost any pattern. A glass ring causes radial angiogenesis around it but none in the centre. “Triangular” injuries to the membrane result in “bunching” of the membrane and radial patterns of angiogenesis. Two such “bunches” placed adjacent to each other cause a papillary pattern of angiogenesis in the intervening tissue. It is not easy to rule out a biochemical stimulus from some intermediary such as distorted epithelium. The similarity of this problem to the problem of angiogenesis in the skin or cornea will be emphasized. 17. A Comparison of the Binding to Erythrocytes of Fibrinogen and Its Plasmin Degradation Products. M. W. RAMPLING AND D. J. R. HENDERSON, Biophysics Department, St Mary’s Hospital
Medical School, London W2 IPG, England A compelling body of data is now available indicating that fibrinogen bound to the erythrocyte membrane can have a significant effect on haemorheology. A study has therefore been undertaken of the factors which affect this binding and, in an attempt to elucidate the binding sites on the molecule involved, fibrinogen degradation products (FDP) produced by plasmin digestion have also been investigated. In all cases the finding was estimated from the partition of iz5I-labelled material between the red cells and suspending phase. It was found that fibrinogen and all the major FDPs bound significantly to erythrocytes, but that the intermediate asymmetric fragment Y did so maximally. The factors affecting the binding were generally similar for all the molecular species investigated. Thus it increased with both decreasing pH (from 8 to 5.4) and with decreasing ionic strength (from 0.2 to 0.1 M) but was independent of temperature (from 4 to 37”) and of calcium ion concentration. Studies using the centrifuge technique have shown that red cell flexibility is influenced by fibrinogen and the large FDPs X and Y, but not by the small late fragments, D and E. Since fibrinogen and fragment X consist essentially of one E and two D fragments covalently linked, while Y consists of one D and one E, the centrifuge results would suggest that at least two widely spaced binding sites are essential if the bound molecules are to affect erythrocyte flexibility. This study was supported by a grant from the Wellcome Trust. and Plasma Fibrinogen in Diabetes. G. D. 0. LOWE, M. SINGARAVELLOO, J. L. S. PARKER, W. G. MANDERSON, C. D. FORBES AND C. R. M. PRENTICE, Department of Medicine and Diabetic Unit, Royal Infirmary, Glasgow G4 OSF, Scotland
18. Platelet Aggregates
Increased plasma fibrinogen levels have been described in diabetics and are associated with vascular complications. Platelet abnormalities are also found in diabetes, and recently increased “circulating” platelet aggregates (CPA) have been detected by formalin fixation in freshly drawn blood. As we have previously found a relationship between CPA and plasma fibrinogen levels (Lowe et al., 1979, Thromb. Res. 14, 377-386), we compared CPA and plasma fibrinogen in 22 diabetics with vascular complications (at least two each of atherosclerosis, peripheral neuropathy, retinopathy, or proteinuria), 20 diabetics without complications, and 42 nondiabetics, matched for age, sex, and smoking habit.
Age (years) Fibrinogen (g/l) CPA (% total) Platelets (x lOgil)
Nondiabetics
Diabetics-no complications
Diabeticscomplications
52 (4)” 2.7 (0.1) 13 (2) 260 (15)
47 (4) 3.2 (0.2) 14 (3) 231 (10)
57 (2) 3.4 (0.2) 25 (3) 254 (14)
c( Mean (+ SEM). Fibrinogen was increased in both groups of diabetics compared with matched controls (P < 0.05). CPA were increased only in diabetics with complications (P < 0.01). There were no significant correlations of CPA with fibrinogen level, platelet count, age, or plasma glucose. The increased CPA may be due to other plasma factors, intrinsic platelet abnormality, or vasculopathy.