Ординатура / Офтальмология / Английские материалы / Retinal Vascular Disease_Joussen, Gardner, Kirchhof_2007

curves have been recorded and that the dye bolus reaching the eye after intravenous injection can clearly be identified. Measurements of arteriovenous passage time (AVP) overcome part of these problems, because it does not depend on the shape of the dye dilution curve. The AVP is defined as the time between the first appearance of the dye in a retinal artery and the corresponding vein (Fig. 9.4). The method, however, requires that the blood of the area supplied by this artery is drained via the adjacent vein. Whether this is true remains to be elucidated. In addition, the appearance of the dye in retinal vessels may be hampered by leakage of fluorescein from retinal vessels in retinal vascular disease or the formation of arteriovenous shunt vessels. The mean dye velocity (MDV) is obtained by measuring fluorescence intensity along two points of a vessel (Fig. 9.5) and provides an estimate of blood flow velocity along the segment of the vessel. Finally, macular blood flow velocities have been quantified by tracking hyperfluorescent and hypofluorescent dots as they pass through perifoveal capillaries assumed to represent leukocytes and erythrocytes,

Fig. 9.4. The arteriovenous passage time (AVP) is calculated from the first appearance of the dye in the artery and the corresponding vein. (From [9])

respectively. This approach, however, does require excellent image quality, because otherwise these hyperfluorescent and hypofluorescent dots cannot be identified unequivocally in consecutive images.

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Fig. 9.5. The time between arrival of the dye at two positions along a vessel can be used to calculate the mean dye velocity (MDV). (From [9])

Based on the acoustical Doppler effect the blood flow velocity in the retrobulbar central retinal artery can be measured using color Doppler imaging (Fig. 9.6). Information on volumetric retinal blood flow cannot be obtained, because the resolution of ultrasound does not allow for measurement of the diameter of the vessel. A variety of parameters is extracted from these measurements including peak systolic flow velocity (PSV), end diastolic flow velocity (EDV) and mean flow velocity (MFV). In addition, a resistive index is calculated as RI = (PSVEDV)/PSV. It is, however, unclear whether the RI represents an adequate measure of retinal vascular

resistance, and factors other than resistance may influence RI as well.

The blue field entoptic technique is based on the blue field entoptic phenomenon, which can be seen best when looking into blue light. Under these conditions many tiny corpuscles can be seen flying around an area of the center of the fovea. This phenomenon is based on the fact that the red, but not white, blood cells absorb short wavelength light. Accordingly, the passage of a white blood cell is perceived as a flying corpuscle. To extract quantitative data from this technique, a simulated particle field is shown to the subject under study. By adjusting the number and the mean velocity of the particles in the simulated particle field with their own perception, an estimate of perimacular white blood cell flux can be extracted as the product of number and mean velocity of the leukocytes. Accordingly, this method is subjective in nature and requires sufficient cooperation from the subject. In addition, leukocyte flux may not be proportional to retinal blood flow in all clinical conditions.

9.3 Blood Flow Regulation

Autoregulation is defined as the intrinsic ability of a vascular bed to change its vascular resistance in response to changes in perfusion pressure. In this very strict sense autoregulation cannot be investigated in humans, because ocular perfusion pressure cannot be modified without affecting other factors such as neural input, circulating hormones and the

metabolic environment. In the retina autoregulation plays an important functional role, because the retina has a constant oxygen demand. Accordingly, intact autoregulation ensures adequate oxygenation of the inner retina during physiological changes in ocular perfusion pressure. Abnormal autoregulation may therefore be detrimental for the eye, resulting in ischemia and hypoxia.

Autoregulatory mechanisms may be more easily investigated in the retina than in many other vascular beds, because the retinal vessels lack autonomic innervation. A decrease in ocular perfusion pressure is normally induced by an experimental increase in intraocular pressure as achieved for instance with a suction cup. This leads to a parallel decrease in ocular perfusion pressure, because the intraocular pressure almost equals the pressure in retinal veins. Using this technique effective autoregulation was evidenced by an adaptation of retinal vascular resistance in face of the decrease in perfusion pressure (Fig. 9.7). Autoregulatory phenomena during an increase in perfusion pressure are inves-

tigated either during a decrease in intraocular perfusion pressure, as achieved for instance after I 9 releasing a suction cup, or during isometric exer-

cise and the concomitant increase in arterial blood pressure. Alternatively, systemic administration of vasoconstrictor substances has been used to study autoregulatory mechanisms in the retina, but this approach has the disadvantage that effects of increased arterial blood pressure cannot necessarily be separated from direct vasoconstrictor effects of the drug. During isometric exercise the retina shows some autoregulatory capacity as evidenced from the plateau in the pressure/flow relationship, with an upper limit of autoregulation of 100 – 110 mm Hg (Fig. 9.8).

Another important aspect of blood flow regulation in the retina is the strong dependence on arterial oxygen tension, with hyperoxia inducing vasodilatation and hypoxia inducing vasoconstriction (Fig. 9.9). This again appears to be related to the constant oxygen demand of the retina. Carbon dioxide is also an important regulator of blood flow, with high-

Fig. 9.8. Retinal pressure/flow curve during isometric exercise. The retinal blood flow is autoregulated up to mean arterial pressures of 100 – 110 mm Hg. (Adapted from [12])

Fig. 9.9. Effects of different mixtures of O2 and N2 on retinal blood flow parameters in healthy subjects as assessed with the HRF. (Adapted from [15])

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Fig. 9.10. Effects of different mixtures of O2 and CO2 on retinal blood flow velocity, retinal vessel diameters and retinal blood flow in healthy subjects. (Adapted from [7])

er arterial CO2 levels leading to vasodilatation and lower levels producing vasoconstriction as in the brain. Accordingly, administration of carbogen (a mixture of CO2 with O2) has been proposed as a therapeutic approach in retinal ischemic and hypoxic disease. It appears, however, that the addition of CO2 to high concentrations of O2 in the inhalate does not sufficiently counteract the pronounced vasoconstrictor effects of hyperoxia (Fig. 9.10).

The retina has the capability to change its metabolic turnover and blood flow in response to neural stimulations. As in the brain this phenomenon is called neurovascular coupling. Flickering light of various frequencies has been used to investigate this phenomenon in some detail. The increase in retinal blood flow following flicker stimulation is in the order of 30 – 40 %, with most of the effect occurring in the smaller vessels. Using high-resolution imaging of retinal vessels, flicker-induced vasodilatation may, however, also be seen in larger vessels (Fig. 9.11). Combining electroretinography with blood flow measurements revealed that the increase in retinal and optic nerve head blood flow after flicker stimulation is closely related to retinal ganglion cell activity.

Fig. 9.11. Flicker responses in retinal arteries during stimulation with 8 Hz (solid circles) and 64 Hz (open triangles) in healthy subjects

Fig. 9.12. Dose dependent effect of the nitric synthase inhibitor L-NMMA on retinal vessel diameter in healthy humans. Data indicate that nitric oxide contributes to physiological tone in the retinal vasculature. (Adapted from [6])

In recent years it has been discovered that the endothelium plays a key role in regulation of retinal vascular tone as it does in other vascular beds. The vascular endothelium produces a number of vasoactive substances including prostacyclin (PGI2) from arachidonic acid, nitric oxide from L-arginine and the potent vasoconstrictor endothelin-1. Nitric oxide plays a key role in the maintenance of vascular tone in the retina, because inhibition of NO synthase with L-arginine analogues causes dose-dependent vasoconstriction (Fig. 9.12). In addition, nitric oxide appears to play a role in several agonist-induced vasodilator effects including histamine, insulin and hypercapnia. Endothelin-1 induces potent vasoconstriction in the human retina with more pronounced effects on the smaller vessels than on the larger vessels. This potent vasoconstrictor effect may be reversed with a specific endothelinA receptor antagonist. Under physiological conditions endothelin-1 appears, however, to contribute little to retinal vascular tone, because administration of an endothelinA

Fig. 9.13. Effect of endothelin-1 and the endothelina receptor antagonist BQ-123 on retinal blood flow parameters. Solid circles endothelin-1 + placebo, solid up triangle placebo + BQ-123, open down triangle endothelin-1 + BQ-123. (Adapted from [8])

receptor antagonist alone has little effect on retinal blood flow (Fig. 9.13). Endothelin-1 has also been shown to play a role in hyperoxia-induced vasoconstriction in the retina.

gated in some detail in patients with diabetes. Vascular abnormalities appear to be an early event in diabetes including venous vasodilatation. There is, however, a number of a contradicting results regarding retinal blood flow in diabetes, which may be related to the different techniques used for the assessment of retinal perfusion parameters. Moreover, it is difficult to investigate retinal blood flow in diabetes under controlled conditions, because both insulin and glucose induce vasodilatation in the eye. There is, however, a variety of studies indicating retinal vascular dysregulation in diabetes. This includes abnormal retinal autoregulation in response to changes in perfusion pressure, altered retinal oxygen reactivity and altered retinal flicker-induced vasodilatation. The alteration in retinal autoregulation in diabetes is more severe in the presence of systemic hypertension, making the retina extremely sensitive to retinal hyperand hypoperfusion. The mechanism underlying changes in retinal oxygen-reactivity in the diabetic retina is largely unclear and may be related to retinal hypoxia, low grade inflammation, endothelial dysfunction, or metabolic changes in the diabetic milieu. Flicker-induced vasodilatation in the diabetic retina is largely reduced, but it is not entirely clear whether this is related to neuronal dysfunction or to alterations in neurovascular coupling. Taken together, alterations of retinal blood flow in diabetes are complex and influenced by a variety of factors (Fig. 9.14). However, due to the lack of longitudinal studies linking these perfusion abnormalities to the progression of diabetic retinopathy, the exact role of retinal vascular dysregulation in the pathophysiology of diabetes-induced alterations in the retina remains obscure.

In clinical routine quantification of retinal blood 9 I flow does not yet play a role in patients with retinal vascular disease. This is related to the fact that all currently available systems for the measurement of retinal blood flow are expensive and time-consum- ing and require significant technical expertise. In addition, large scale studies clearly linking alterations in retinal blood flow to the development and progression of retinal vascular disease are lacking. Accordingly, new techniques are required for the quantification of retinal ischemia and hypoxia in humans to gain more insight into the physiopatholo-

gy of retinal vascular disease.

References

1.Alm A, Bill A (1973) Ocular and optic nerve blood flow at normal and increased intraocular pressures in monkeys (Macaca irus): a study with radioactively labelled microspheres including flow determinations in brain and some other tissues. Exp Eye Res 15(1):15 – 29

2.Cioffi GA, Granstam E, Alm A (2003) Ocular circulation. In: Kaufman PL, Alm A (eds) Adler’s physiology of the eye, 10th edn. Mosby, St. Louis, pp 747 – 784

3.Delaey C, van de Voorde J (2000) Regulatory mechanisms in the retinal and choroidal circulation. Ophthalmic Res 32:249 – 56

4.Haefliger IO, Flammer J, Beny JL, Luscher TF (2001) Endo- thelium-dependent vasoactive modulation in the ophthalmic circulation. Prog Retin Eye Res 20:209 – 225

5.Harris A, Chung HS, Ciulla TA, et al. (1999) Progress in measurement of ocular blood flow and relevance to our understanding of glaucoma and age-related macular degeneration. Prog Retin Eye Res 18:669 – 687

6.Huemer K-H, Garhöfer G, Zawinka C, Golestani E, Litschauer B, Schmetterer, Dorner GT (2003) Effects of dopamine on human retinal vessel diameter and its modulation during flicker stimulation. Am J Physiol Heart Circ Physiol 284:358 – 363

7.Luksch A, Garhöfer G, Imhof A, Polak K, Polska E, Dorner GT, Anzenhofer S, Wolzt M, Schmetterer L (2002) Effect of

inhalation of different mixtures of O2 and CO2 on retinal blood flow. Br J Ophthalmol 86:1143 – 1147

8.Polak K, Luksch A, Frank B, Jandrasits K, Polska E, Schmetterer L (2003) Regulation of human retinal blood flow by endothelin-1. Exp Eye Res 76:633 – 640

9.Rechtman E, Harris A, Kumar R, Cantor LB, Ventrapragada S, Desai M, Friedman S, Kageman L, Garozzi HJ (2003) An update on retinal circulation assessment technologies. Curr Eye Res 27:329 – 343

10.Riva CE, Grunwald JE, Petrig BL (1986) Autoregulation of human retinal blood flow. Invest Ophthalmol Vis Sci 27:1706 – 1712

11.Riva CE, Logean E, Falsini B (2005) Visually evoked hemodynamical response and assessment of neurovascular coupling in the optic nerve head and retina. Prog Retin Eye Res 24:183 – 215

12.Robinson F, Riva CE, Grunwald JE, Petrig Bl, Sinclair SH (1986) Retinal blood flow autoregulation in response to an acute increase in blood pressure. Invest Ophthalmol Vis Sci 27:722 – 726

13.Schmetterer L, Polak K (2001) Role of nitric oxide in the control of ocular blood flow. Prog Ret Eye Res 20:823 – 847

14.Schmetterer L, Wolzt M (1999) Ocular blood flow and associated functional deviations in diabetic retinopathy. Diabetologia 42:387 – 405

15.Strenn K, Menapace R, Rainer G, Findl O, Wolzt M, Schmetterer L (1997) Reproducibility and sensitivity of scanning

laser Doppler flowmetry during graded changes in PO2. Br J Ophthalmol 81:360 – 364

Core Messages

Gene therapy is a process whereby specific genes are delivered into cells to restore a missing function, alter an aberrant function or give cells a new function

There are two approaches to gene delivery: ex vivo (the introduction of the genetic material occurs in cells that have been removed from the body) and in vivo (genes are delivered to cells inside the body)

Gene therapy can target both somatic and germ line cells, although somatic cells have been the primary focus of most therapeutic protocols

There are a variety of gene delivery methods, ranging from the simple incubation of target cells with naked DNA to complex biological systems such as genetically engineered viruses

A myriad of retinal disease and pathologies may benefit from these approaches

In a limited number of clinical trials, gene therapy for ocular disease has proven safe. Compartmentalization of the eye, relative immune isolation and the characteristics of the terminally differentiated intraocular cells may be important factors in the relative safety of these approaches

10.1.1 Introduction

Development of novel molecular biology techniques in the 1970s and 1980s furnished scientists with new tools to advance the study and treatment of human disease. Progress in the understanding of bacterial and viral biology led to innovations in molecular cloning and chimeric plasmid construction. Advances in nucleic acid sequencing allowed researchers to gain a better understanding of genes and the ability to study gene mutations. Unraveling the minutiae of molecular events involved in gene transcription and translation furthered the analysis of cellular pathways and their complex interrelationships. Production of proteins ex vivo allowed physicians to treat diseases such as diabetes with synthetic human insulin, ending the dependency on animal sources.

These advances enabled researchers to propose a new question: can an inherited disorder and/or pathological event be treated at the genetic level? This question introduced the science of gene therapy. Gene therapy was initially an attempt to replace defective genes with functional ones. Investigators believed this was the best (and perhaps only) way to treat rare, genetically well-defined, loss-of-function diseases. The first gene therapy trial commenced in 1990, was

directed by W. French Anderson and Michael Blaese and was designed to treat a severe combined immunodeficiency syndrome (SCID) caused by a deficiency of the enzyme adenosine deaminase (ADA) [1]. Since then more than 350 somatic cell gene therapy trials have been conducted or are underway. Although gene therapy holds considerable promise, ethical and safety issues are important to consider. Sadly, despite extensive pre-clinical studies, severe complications have been observed in at least two different human clinical trials [6, 7]. While currently there are no commercially available gene therapy-based medications, as safe and effective drugs are developed a variety of disease processes are likely to be amenable to this form of therapy.

10.1.2 Delivery Systems

Essentials

Viral, non-viral and physical methods exist for gene transfer

Expression efficiency can vary greatly depending on the chosen vehicle

All delivery systems have certain advantages and disadvantages (see Table 10.1.1)

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10.1.2.1 Retroviral (Fig. 10.1.1)

Retroviruses were the first virus-mediated means of gene transfer. The original vectors were developed from oncoretroviruses such as Molony murine leukemia virus, Rous sarcoma virus, spleen necrosis virus, and avian leukosis virus [3]. These offered suitable integrative gene expression but are unable to infect non-dividing cells. Retroviruses that were capable of infecting both proliferating and stationary cells, like those of the retina, became a desirable alternative. Identification and study of the lentivirus HIV-1 led to research for its use in gene transfer methods. Lentiviral vectors have many advantages when compared to other retroviral vectors, such as large carrying capacity, ease of production/purification of high titer particles and less likelihood of integrating into a spot that will cause deleterious mutations [27]. In 2003, the first human clinical trial using a HIV-1 based lentiviral vector (VRX496) to treat HIV-positive patients was initiated [19].

Lentiviruses are single-stranded RNA viruses 80 – 130 nm in size with an approximate 9.2 kb

genome [14]. The RNA is packaged within a viral core surrounded by an envelope, derived from the host’s cell membrane. The envelope contains viral glycoproteins used for binding cellular receptors.

Lentiviral vectors have undergone several generations of construction and manipulations in the laboratory. Each version has increased the vectors’ safety of use and transgene expression in vivo and in vitro.

10.1 Gene Therapy for Proliferative Ocular Disease

Current vectors are considered self-inactivating (SIN) due to deletions in the 3’ long terminal repeat (LTR), rendering native viral promoters inactive [13]. Implementation of several enhancing factors such as the central polypurine tract (cPPT) and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) have increased transgene uptake and expression stability, respectively [22].

10.1.2.2 Adenovirus (Fig. 10.1.2)

Adenoviruses were first used for gene transfer in 1985 [2]. Since then, they have been used in hundreds of gene therapy experiments.

Adenoviruses are non-enveloped icosahedral viral particles approximately 80 nm in size. The genome consists of double stranded DNA, 36 kb in length [17].

The wild type genome is transcribed in two nonexclusive phases labeled early and late. Early gene products are involved in cell cycle hijacking, immune response evasion, and replication. Late genes are responsible for viral assembly and packaging [21].

Recombinant adenoviral vectors are produced replication deficient. This is accomplished by removing the “early” expressing genes. Current vectors are considered gutless or lacking all viral genes, retaining only cis-acting elements such as the inverted terminal repeats (ITRs). This strategy introduces the necessity to produce viral particles in a helper dependent manner. Genes

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required for structural components and replication are provided in trans via co-transfection with plasmids containing necessary genes or with packaging cell lines that stably produce required components.

10.1.2.3 AAV (Fig. 10.1.3)

The use of adeno-associated viral (AAV) vectors in gene transfer has gained ever-increasing popularity in the last 10 years. AAV is now a well-studied vehicle for viral mediated gene transfer into ocular tissue.

AAV is a small icosahedral single stranded DNA virus, ranging 20 – 25 nm in size. Its genome is small, composed of 4.7 kb of DNA with two open reading frames (ORF) (coding for the Rep and Cap genes) [18].

Recombinant AAV is considered gutless retaining only the inverted tandem repeats (ITR). First-generation AAV vectors retained the Rep gene (involved with integration events) but it was later removed to increase vector carrying capacity and is usually sup-