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defined as sympathovagal balance but that couldas well be defined as excitation-inhibition bal-ance. Hence, on the one hand, a new way ofthinking seems necessary to address a conceptthat can be only partly quantified, just like intel-ligence, stress, or homeostasis. On the otherhand, only future research will establish howmuch the study of a patterned rhythmic code willrepresent a useful approach to complexity.

References

1. Akselrod, S., D. Gordon, F. A. Ubel, D. C. Shannon, andR. J. Cohen. Power spectrum analysis of heart rate fluctu-ation: a quantitative probe of beat-to-beat cardiovascularcontrol. Science 213: 220222, 1981.

2. Eckberg, D. L. Sympathovagal balance. A criticalappraisal. Circulation 96: 32243232, 1997.

3. Jasson, S., C. Mdigue, P. Maison-Blanche, N. Montano,L. Meyer, C. Verneiren, P. Mansier, P. Coumel, A.Malliani, and B. Swynghedauw. Instant power spectrumanalysis of heart rate variability during orthostatic tiltusing a time-/frequency-domain method. Circulation 96:35213526, 1997.

4. Malliani. A. Cardiovascular sympathetic afferent fibers.Rev. Physiol. Biochem. Pharmacol. 94: 1174, 1982.

5. Malliani, A. Association of heart rate variability compo-nents with physiological regulatory mechanisms. In:Heart Rate Variability, edited by M. Malik and A. J.Camm. Armonk, NY: Futura, 1995, p. 173188.

6. Malliani, A. The autonomic nervous system: a Sherring-tonian revision of its integrated properties in the controlof circulation.J. Auton. Nerv. Syst. 64: 158161, 1997.

7. Malliani, A., M. Pagani, R. Furlan, S. Guzzetti, D. Lucini, N.

Montano, S. Cerutti, and G. S. Mela. Individual recognitionby heart rate variability of two different autonomic profilesrelated to posture. Circulation 96: 41434145, 1997.

8. Malliani, A., M. Pagani, F. Lombardi, and S. Cerutti. Car-diovascular neural regulation explored in the frequencydomain. Circulation 84: 482492, 1991.

9. Montano, N., T. Gnecchi Ruscone, A. Porta, F. Lombardi,M. Pagani, and A. Malliani. Power spectrum analysis ofheart rate variability to assess the changes in sympatho-vagal balance during graded orthostatic tilt. Circulation90: 18261831, 1994.

10. Pagani, M., F. Lombardi, S. Guzzetti, O. Rimoldi, R. Furlan,P. Pizzinelli, G. Sandrone, G. Malfatto, S. DellOrto, E. Pic-caluga, M. Turiel, G. Baselli, S. Cerutti, and A. Malliani.Power spectral analysis of heart rate and arterial pressurevariabilities as a marker of sympathovagal interaction inman and conscious dog. Circ. Res. 58:178193, 1986.

11. Pagani, M., N. Montano, A. Porta, A. Malliani, F. M.Abboud, C. L. Birkett, and V. K. Somers. Relationshipbetween spectral components of cardiovascular variabil-ities and direct measures of muscle sympathetic nerveactivity in humans. Circulation 95: 14411448, 1997.

12. Piepoli, M., P. Sleight, S. Leuzzi, F. Valle, G. Spadacini, C.Passino, J. Johnston, and L. Bernardi. Origin of respiratorysinus arrhythmia in conscious humans: an important rolefor the arterial carotid baroreceptors. Circulation 95:

18131821, 1997.13. Schwartz, P. J., M. Pagani, F. Lombardi, A. Malliani, and

A. M. Brown. A cardio-cardiac sympatho-vagal reflex inthe cat. Circ. Res. 32: 215221, 1973.

14. Sherrington, C. S. The Integrative Action of the NervousSystem. New Haven: Yale Univ. Press, 1906.

15. Tougas, G., M. Kamath, G. Watteel, D. Fitzpatrick, E. L.Fallen, R. H. Hunt, and A. R. Upton. Modulation of neu-rocardiac function by esophageal stimulation in humans.Clin. Sci. 92: 167174, 1997.

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The Role of the Sinusoidal Endothelium

in Liver FunctionJrg Reichen

Microvascular exchange in the liver is governed by fenestrations in sinusoidalendothelial cells and can be manipulated pharmacologically. Microvascularexchange is affected in alcoholic liver disease and cirrhosis, the formerleading to a loss of fenestrae, the latter to sinusoidal capillarization andthereby to loss of liver function in disease.

The endothelium of the liver permits free

access to proteins and protein-bound sub-strates from the sinusoid to the space of Disse andthereby to the hepatocytes, owing to the presenceof fenestrae arranged in sieve plates (Fig. 1). Sinu-soidal fenestrae average 175 nm in diameter andoccupy ~68% of the sinusoidal surface area (forreview, see Ref. 15). There is no basal lamina,

allowing free passage of macromolecules up to

medium-sized chylomicrons (15). This featureexplains the fact that the liver can extract a vari-ety of tightly protein-bound endo- and xenobi-otics. The fenestrae are surrounded by a densering of actin, whereas the sieve plates are formedby microtubules. Both number and size of fenes-trae can be regulated by a variety of processesthat will impact on hepatic function.

Different techniques have been used to assessthe function of the sinusoidal fenestrations,including the multiple-indicator dilution tech-

J. Reichen is Professor of Medicine in the Department ofClinical Pharmacology of the University of Berne, Murten-strasse 35, 3010 Berne, Switzerland.

The fenestrae aresurrounded by adense ring ofactin....

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nique (5), in vivo microscopy (10), stereologicalanalysis of scanning electron micrographs (15),atomic force microscopy (2), and digitized imageanalysis using actin staining (4).

Among the different techniques listed above,only the multiple-indicator dilution and intravi-tal microscopy are apt to probe microvascularexchange in vivo; only in vivo methods are suit-able to investigate pathophysiological changesin different forms of liver disease. These twomethods are complementary: the multiple-indi-cator dilution technique permits a global assess-

ment of microvascular exchange and even oftransport and enzymatic processes (6), whereasintravital microscopy can visualize a single sinu-soid and the interaction of blood flow with thecellular constituents of the sinusoid. This tech-nique has been instrumental in understandingthe effects of inflammation, reperfusion, andtoxic injury. The reader is referred to a recentarticle by one of the pioneers in the field for fur-ther details (10). In the following section onlythe multiple-indicator dilution technique is con-sidered further.

News Physiol. Sci. Volume 14 June 1999118

FIGURE 1. Scanning electron micrograph of a perfusion-fixed liver. The endothelial cells with their typical fenestrations abovea hepatocyte with its microvilli protruding into the space of Disse are shown.

FIGURE 2. Multiple-indicator dilution curves obtained in a normal (A) and a cirrhotic (B) in situ perfused rat liver. Hepaticvenous outflow curves (given as natural logarithm of its frequency functions) of erythrocytes (), albumin (), and sucrose ()are shown. InA, the flow-limited pattern expected in normal liver is shown, whereas the pattern in B is diffusion limited (7).From Ref. 12 with permission.

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The multiple-indicator dilution is based on avery old physiological principle using indicators

to calculate flow and volume of distributionaccording to Stewart in 1894 and Hamilton in1929 (see Ref. 1) and was introduced in its pres-ent form in 1955 by Chinard and colleagues (forreferences and theoretical basis, see Ref. 1). Theexperimental approach is very simple: a mixtureof indicators distributing into sinusoids (e.g., ery-throcytes), the extravascular space (e.g., albuminor sucrose), and the intracellular space (e.g., ureaor water) are injected as a bolus into the feeding

vessel (in the case of the liver, the portal vein);immediately thereafter, the outflow is sampled at

short time intervals. Such studies have been per-formed in the intact organism, including humans(9). To gain the maximum information, however,studies in the isolated organ such as the in situperfused rat liver are preferable because in thispreparation, recirculation of the indicator is pre-vented. A typical experiment is shown in Fig. 2A.It shows the typical, flow-limited patternexpected in a capillary with free exchange ofmatter between sinusoidal lumen and the

News Physiol. Sci. Volume 14 June 1999 119

FIGURE 3. Correlation between the extravascular space available to albumin (EVA) and hepatic function, measured as N-demethylation of aminopyrine by a breath test (ABT) in cirrhotic rat liver. In multivariate analysis, EVA was found to be the maindeterminant of hepatic function in cirrhotic rat. From Ref. 11 with permission.

FIGURE 4. Multiple-indicator dilution curves in cirrhotic rat liver in the basal state, during administration of endothelin 1 (10 -9

mol/l; ET) and during coadministration of endothelin at the same concentrations + the mixed antagonist bosentan (10 -5 mol/l).The symbols are as in Fig. 2. Endothelin leads to further impediment to microvascular exchange, as judged from the closer super-imposition of the albumin on the erythrocyte curve. This is reversible by administering the mixed antagonist; as a matter of fact,microvascular exchange was improved significantly. From Ref. 12 with permission.

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extravascular space (in this case the space ofDisse): the erythrocytes appear earlier and peakhigher than both extravascular labels, in this casealbumin and sucrose. The reason for this is evi-dent: the extravascular labels distribute into alarger space than the erythrocytes. The latteradvance with flow before diffusion of the indica-tors back into the vascular space occurs: theiroutflow curves are damped and delayed com-

pared with those of the intravascular label.According to Goreskys concept, these curvescan be brought to superimposition by correctingoutflow times and concentrations for the ratio ofintra- to extravascular space. The small differencebetween the albumin and sucrose curves iscaused by molecular sieving in the space ofDisse: the albumin behaves like an excludedmolecule, whereas sucrose, owing to its muchsmaller size, diffuses into a larger space of theextracellular matrix.

Quite a different picture emerges when thisexperiment is performed in the liver of a cirrhotic

rat (Fig. 2B): now the albumin curve is virtuallysuperimposed upon the erythrocyte curve,whereas the sucrose curve demonstrates a breakfrom its normal, monotonous decay. This corre-sponds to a diffusion-limited pattern as seen, forexample, in myocardium with an endotheliumwithout fenestrations and a basement membrane(7). If one takes the calculated extravascularspace accessible to albumin (EVA) as a measureof sinusoidal capillarization, there is excellentcorrelation with different aspects of hepatic func-tion (Fig. 3), EVA being the main determinant ofliver function in cirrhosis (11). This may be ofclinical relevance because hepatic failure is themain reason for death in cirrhosis and, con-versely, hepatic residual function, measured indifferent ways, is a main predictor of survival.

This alteration of hepatic microvascularexchange is caused not only by sinusoidal capil-larization but, at least in the case of alcoholic liverdisease, also by a loss of fenestrae (8). Finally, it isconceivable that there are humoral factors affect-ing the fenestrae be it size or numbers. Thusendothelin 1 (12), serotonin (4), endotoxin, andnicotine (reviewed in Ref. 15) decrease the num-

ber of sinusoidal fenestrae, whereas acute alcoholadministration, pressure, and microfilament-dis-rupting agents increase them (15).

The finding that pharmacological agents areable to alter the structure of sinusoidal endothe-lial cells has opened novel avenues for treatmentof portal hypertension and has raised the hopethat by altering microvascular exchange onecould improve liver function. Indeed, wedemonstrated that the calcium antagonist vera-pamil improved microvascular exchange in the

cirrhotic rat liver (14) and thereby amelioratedhepatic clearance and function (13, 14). In thiscase it remained unclear whether this wasindeed caused by alterations in the numberand/or size of sinusoidal fenestrations or by theselection of sinusoids with better microvascularexchange characteristics. The case appears muchclearer for endothelin 1; this potent vasoconstric-tor induces a reversible inhibition of microvascu-

lar exchange in normal mouse and rat liver thatis best explained by constriction of the fenestrae(3, 12). In cirrhotic rat liver, endothelin 1 furtherimpedes the already impaired access of albuminto the extravascular space (Fig. 4) whereas themixed antagonist bosentan amelioratesmicrovascular exchange significantly abovebaseline (12). These experiments largely favor thecontention that even in chronic liver diseasemicrovascular exchange in the hepatic endothe-lium is amenable to pharmacological manipula-tion. Eventually, this might lead to treatments thatnot only prevent the consequences of portal

hypertension, such as variceal bleeding, butmight also improve liver function, the maindeterminant of survival in patients with end-stageliver disease.

The author thanks Prof. O. Mller and his staff from theDepartment of Anatomy at the University of Berne for thescanning electron micrograph shown in Fig. 1.

This work was supported by a grant from the SwissNational Foundation for Scientific Research (No.3245349.95).

References

1. Bassingthwaighte, J. B., and C. A. Goresky. Modeling inthe analysis of solute and water exchange in the microvas-culature. In: Handbook of Physiology. The CardiovascularSystem. Microcirculation. Bethesda, MD: Am. Physiol.Soc., 1984, sect. 2, vol. IV, pt. 1, p. 549626.

2. Braet, F., W. H. J. Kalle, R. B. De Zanger, B. G. DeGrooth, A. K. Raap, H. J. Tanke, and E. Wisse. Compara-tive atomic force and scanning electron microscopy: aninvestigation on fenestrated endothelial cells in vitro.J.Microsc. 181: 1017, 1996.

3. Castillo, M. B., M. W. Berchtold, T. Rlicke, B. Schwaller,V. Gotzos, M. Pinzani, J. Reichen, and M. R. Celio.Ectopic expression of the calcium-binding protein par-valbumin in mouse liver endothelial cells. Hepatology25: 11541159, 1997.

4. Gatmaitan, Z., L. Varticovski, L. Ling, R. Mikkelsen, A. M.Steffan, and I. M. Arias. Studies on fenestral contractionin rat liver endothelial cells in culture.Am. J. Pathol. 148:20272041, 1996.

5. Goresky, C. A. A linear method for determining liver sinu-soidal and extravascular volume. Am. J. Physiol. 204:626640, 1963.

6. Goresky, C. A., E. R. Gordon, and G. G. Bach. Uptake ofmonohydric alcohols by liver: demonstration of a sharedenzymic space. Am. J. Physiol. 244 (Gastrointest. LiverPhysiol. 7): G198G214, 1983.

7. Goresky, C. A., W. H. Ziegler, and G. G. Bach. Capillaryexchange modelling. Barrier-limited and flow-limited

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. . .the albuminbehaves like anexcludedmolecule. . . .

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distribution. Circ. Res. 27: 739764, 1970.8. Horn, T., J. Junge, and P. Christoffersen. Early alcoholic

liver injury: changes of the Disse space in acinar zone 3.Liver 5: 301310, 1985.

9. Huet, P. M., C. A. Goresky, J. P. Villeneuve, D. Marleau,and J. O. Lough. Assessment of liver microcirculation inhuman cirrhosis.J. Clin. Invest. 70: 12341244, 1982.

10. McCuskey, R. S., and F. D. Reilly. Hepatic microvascula-ture: dynamic structure and its regulation. Semin. LiverDis. 13: 112, 1993.

11. Reichen, J., B. Egger, N. Ohara, T. B. Zeltner, T. Zysset,

and A. Zimmermann. Determinants of hepatic functionsin liver cirrhosis in the rat: a multivariate analysis.J. Clin.Invest. 82: 20692076, 1988.

12. Reichen, J., A. L. Gerbes, M. J. Steiner, H. Sgesser, and

M. Clozel. The effect of endothelin and its antagonistBosentan on hemodynamics and microvascularexchange in cirrhotic rat liver. J. Hepatol. 28:10201030, 1998.

13. Reichen, J., A. Hirlinger, H. R. Ha, and H. Saegesser.Chronic verapamil administration lowers portal pressureand improves hepatic function in rats with liver cirrhosis.J. Hepatol. 3: 4958, 1986.

14. Reichen, J. and M. Le. Verapamil favorably influenceshepatic microvascular exchange and function in rats withcirrhosis of the liver.J. Clin. Invest. 78: 448455, 1986.

15. Wisse, E., F. Braet, D. Luo, Z.R. De, D. Jans, E. Crabbe,and A. Vermoesen. Structure and function of sinusoidallining cells in the liver. Toxicol. Pathol 24: 100111,1996.

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Arteriogenesis Versus Angiogenesis: TwoMechanisms of Vessel GrowthI. Buschmann and W. Schaper

After birth, new blood vessel formation proceeds via angiogenesis or arteriogenesis.Angiogenesis (capillary sprouting) results in higher capillary density.Arteriogenesis (rapid proliferation of collateral arteries) is potentially able tosignificantly alter the outcome of coronary and peripheral artery disease.The processes share some growth features but differ in many aspects.

The formation of a functional, integrated vas-cular network is a fundamental process in thegrowth and maintenance of tissue. Vasculariza-tion occurs by three distinct processes: vasculo-genesis, angiogenesis, and arteriogenesis.

Vasculogenesis is the earliest morphogeneticprocess of vascular development and takes placeexclusively during the early embryonic stages.Indeed, the cardiovascular system is the firstorgan system that is laid down during embryonicdevelopment. Vasculogenesis consists of the dif-ferentiation of angioblasts (the precursors ofendothelial cells) into blood islands, which thenfuse to form primitive capillary plexuses (2). Theplexuses subsequently grow by angiogenesis(sprouting and tube formation by single endothe-lial cells within a preexisting capillary plexus),invade target tissues, and give rise to the primi-

tive vascular system of embryonic organs. Whenthe heart starts beating (after 2 days in the chickembryo) and the circulation commences shortlythereafter, significant changes occur in the mor-phogenesis of the vascular system. Some vesselsof the primary plexus remain as capillaries,

whereas others differentiate into arteries or veins(10). Although regression and differentiation arestill unknown processes, it is believed that hemo-dynamic forces play a central role. Cessation ofblood flow into a capillary segment causes theregression of the vessel, whereas an increase inpressure and shear stress may be an inductivefactor for the local recruitment of smooth musclecells, leading to the differentiation of a capillaryvessel into an artery or vein. The adult vascula-ture, with a surface area of ~1,000 m2, finallyconsists of large arteries, internally lined byendothelial cells and well ensheathed by smoothmuscle cells, that progressively branch intosmaller and smaller vessels, terminating in pre-capillary arterioles that then give rise to capillar-ies. These vascular tubes are comprised almostentirely of endothelial cells that are only in a few

cases coated by a smooth muscle cell like thepericyte. The capillaries then feed into postcapil-lary venules that progressively associate intolarger and larger venous structures (15).

The term angiogenesis was introduced in 1935by Hertig to describe the formation of new bloodvessels in the placenta and, later, in 1971, byFolkman to describe the neovascularizationaccompanying the growth of solid tumors. Angio-genesis is a process by which new capillary bloodvessels sprout from a preexisting blood vessel

I. Buschmann and W. Schaper are in the Department forExperimental Cardiology of the Max Planck Institute forPhysiological and Clinical Research, Benekestrasse 2, D-61231 Bad Nauheim, Germany.

Vasculogenesisconsists of thedifferentiation ofangioblasts.. . .