UNIVERSITY OF LONDON OBJECTIVE ASSESSMENT OF MICROCIRCULATORY RESPONSE IN VENOUS DISEASE TO THERAPY A Thesis Submitted To University College London In Fulfilment of the Requirements of the Master of Surgery (M.S.) by Syed Sulaiman Shoab MBBS, FCPS, FRCSI, FRCS (General Surgery) 2009 Department Of Surgery U.C.L. Medical School and U.C.L. Hospitals The Middlesex Hospital, London-UK ABSTRACT Venous stasis is associated with leucocyte & endothelial activation as well as local growth factor response in chronic venous distasease (CVD). There is no good data on the response of this increased activation to treatment. The theme of this thesis was to show changes in leucocyte/endothelial activation as well as microcirculatory stasis in response to medical, surgical and compression therapy. Thus these may act as objective measures of response to treatment. Vascular endothelial growth factor (VEGF) is being investigated extensively in various arterial scenarios I demonstrated high plasma levels of among patients with CVD for the first time. Levels in patients were about 60% higher (82pg Vs 52 pg in controls). This may represent an (reparative!) angiogenic response existing along with the leucocyte inflammatory response. I used a model of medical treatment (60 days oral flavonoid therapy) and demonstrated significant change in plasma VEGF (50% reduction i.e. 98 pg to 57 pg/dl), ICAM (32%), VCAM (29%) & lactoferrin (36%) levels in patients. Thus I showed that endothelial cell activation (ECA) as well as VEGF might be used as an objective surrogate marker in CVD. I propose that amelioration of endothelial activation may be a mechanism of action for these compounds. I studied the response of these parameters to surgical treatment of varicose veins in 20 patients. I showed that there is an increased plasma lactoferrin at 4 weeks that goes below starting base line levels at 6 months (865 Vs 870 Vs 519). VEGF levels continued to increase (65 Vs 83 Vs 134 pg/dl) in these patients and this may represent vascular remodelling. Although not all of them are easily explainable, the microcirculatory parameters were shown to have a definable response to therapy. I used a new apparatus (Laser capillary anemometer) to assess the response of the velocity of blood in the microvasculature of patients with venous, arterial or mixed disease. I demonstrated that compression increases velocity of blood in the sub-papillary plexus & lower levels of compression (20 mm Hg) are more effective in increasing velocity in patients with mixed disease. This may explain the basis for using compression therapy in these patients. 2 Thus I have shown that various parameters of microcirculation may be used to assess the response to therapy in CVD. Future uses of these findings may include design of new and novel therapeutic approaches and to prognosticate for the development of skin changes and ulceration of the leg in CVD. 3 Figure 1 Illustrates both the leucocyte priming as well as presence of factors in the serum that mediate it in CVD. Micrographs of polymorphonuclear granulocytes showing tetrazolium crystal formation after nitroblue tetrazolium reduction (A,B,C) and projection of pseudopods (D,E,F). Control blood of individuals without symptoms (A,D), patient whole blood (B,E), and native granulocytes incubated in patient plasma (C,F). Bar represents 10 m. Magnification is same for all patients. From Takase S et al with permission.1 4 Figure 2 depicts the interaction between the leucocytes and the endothelium that has become so important in the understanding of tissue damage in CVD. The reduced arterio-venous pressure gradient (PA-PV) due to lower flow rates leads to leucocyte margination (decreased shear/ physical characteristics of the leucocyte). This leads to increased cell adhesion molecule expression. There is initial weak adhesion followed by stronger adhesion. Different adhesion molecules are involved in these processes. These leucocytes eventually diapedese. These activated leucocytes may cause tissue damage locally either directly or by cytokine release triggering a chemotactic reaction for other white cells. The adhesion molecule expression has been used in many of the studies that are part of this thesis (From Hunt & Jurd 1998, by permission).60 ………………………………….. 5 CONTENTS OBJECTIVE ASSESSMENT OF MICROCIRCULATORY RESPONSE IN VENOUS DISEASE TO THERAPY...................................................................................................... 1 Statement of Originality ...................................................................................................... 8 Acknowledgments................................................................................................................ 10 PART I ................................................................................................................................. 11 INTRODUCTION ............................................................................................................... 11 1.1 DEVELOPMENT OF THE UNDERSTANDING OF CVD ................................................................. 13 1.2 EPIDEMIOLOGY OF CVD................................................................................................................. 18 INHERITANCE .......................................................................................................................................... 24 1.3 Applied Venous Physiology of the Lower Limbs.......................................................... 24 THE CALF MUSCLE PUMP & ORTHOSTATIC VENOUS PRESSURES .................... 25 Other Aspects of the Venous Circulation............................................................................. 27 1.5 BIOLOGY OF CHRONIC VENOUS DISEASE....................................................................................... 28 1.7 ADHESION MOLECULES IN CVD..................................................................................................... 36 1.8 INVESTIGATION OF CHRONIC VENOUS DISEASE ............................................................................ 39 F= REFLECTED FREQUENCY .................................................................................................................... 41 INFLAMMATORY MEDIATORS ................................................................................................................. 58 EFFECT ON CYTOKINE RELEASE ............................................................................................................. 58 EFFECTS ON VENOUS WALL COMPOSITION .............................................................................................. 59 1.10 COMPRESSION THERAPY - MECHANISM OF.................................................................................. 66 ACTION & THE MICROCIRCULATION.................................................................................................... 66 1.11 SURGICAL/ INTERVENTIONAL TREATMENT OF ............................................................................ 72 CHRONIC VENOUS DISEASE .................................................................................................................. 72 1.12 REPORTING STANDARDS OF OUTCOME........................................................................... 77 PART II................................................................................................................................ 79 HYPOTHESIS ..................................................................................................................... 79 PART III- ............................................................................................................................. 82 METHODOLOGY............................................................................................................... 82 3.1 PROTOCOL FOR QUANTIFYING LOWER LIMB VENOUS REFLUX USING DUPLEX ULTRASOUND SCANNING .............................................................................................................................................. 83 Q = TAV X AREA X T ........................................................................................................................... 85 PROTOCOL FOR VENOUS REFLUX TEST BY PHOTOPLETHYSMOGRAPHY (PPG)................................... 86 PROTOCOL FOR MEASURING RESTING ANKLE BRACHIAL PRESSURE INDICES (ABPI)........................ 86 Data Analysis ....................................................................................................................... 87 3.1.1 HUMAN SOLUBLE ICAM-1 IMMUNOASSAY ................................................................................. 87 PRINCIPLE OF THE PROCEDURE .............................................................................................................. 97 3.3 LASER DOPPLER ANEMOMETER FOR MEASURING BLOOD CELL............................................... 100 VELOCITIES IN PERPENDICULAR CAPILLARY LOOPS ....................................................................... 100 PART IV ............................................................................................................................ 108 6 STUDIES ........................................................................................................................... 108 4.1 VASCULAR ENDOTHELIAL GROWTH FACTOR.............................................................................. 109 (VEGF) IN CVD .................................................................................................................................