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The scar neovasculature after myocardial infarction in rats

Departments of ¹Mechanical Engineering and ²Radiation Oncology, Temple University, Philadelphia, Pennsylvania; and Divisions of ³Cardiovascular Diseases and ⁴Rheumatology, ⁵Department of Biomedical Engineering, University of Tennessee Health Science Center, Memphis, Tennessee

Submitted 4 January 2005 ; accepted in final form 17 February 2005

	ABSTRACT

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A series of novel techniques, adapted from the field of tumor biology, were developed to quantify vascular structure and function and to explore the role of ANG II receptor AT1 in cardiac remodeling after myocardial infarction (MI). We examined the scar neovasculature at 1–4 wk post-MI in Sprague-Dawley rats with a view toward its ability to deliver and exchange oxygen. CD31 and DiOC₇(3) staining was used to visualize anatomical vessels vs. those perfused. EF5/Cy3 immunohistochemical staining was used to quantify tissue hypoxia. We compared untreated controls with rats treated with losartan, an AT1 receptor antagonist. Our findings indicated that, at the infarct site, there was not only a 42–75% (1–4 wk post-MI) decrease in the number of anatomical vessels compared with controls but also a decrease in the fraction of perfused vessels from 70% in normal coronary vasculature to 48% at the infarct site. These changes were accompanied by progressive increases in diffusion distance and tissue hypoxia (100% increase in EF5/Cy3 staining at 4 wk post-MI). Losartan-treated rats exhibited a significantly less marked reduction in vascular perfusion and a significantly lesser extent of tissue hypoxia. Over the course of 4 wk post-MI, there is a reduction in coronary vasculature at the infarct site, the extent of which is attenuated by losartan. These findings implicate AT1 receptor upregulation, and perhaps angiotensin-related peptides, as being antiangiogenic.

hypoxic marker; perfusion; myocardial infarction; microcirculation

In rats with a transmural MI, a significant reduction in coronary vascular density and diffusion distance in the infarct site has been reported (1, 6, 7, 12, 13, 25, 37). Few studies have addressed the scar neovasculature, its vascular composition, its capacity to deliver oxygen, and whether regions of hypoxia are present after MI. Although a recent study has shown that levels of hypoxia [as indicated by hypoxia-inducible factor 1 alpha (HIF-1) upregulation] in the infarcted myocardium of patients with ischemic cardiomyopathy are increased (24), the relationship between variables such as vascular composition and tissue hypoxia in the scar has not been clearly established in part because these variables have not been measured in the same sections of the tissue.

ANG II, produced de novo by macrophages and myofibroblasts at the site of MI, has auto/paracrine properties that contribute to tissue repair. Integral to its auto/paracrine properties is the expression of angiotensin receptors by these cells, which in rats are predominantly of the AT1 subtype (17, 31). The regulatory role of ANG II on cardiac remodeling has been well studied. After infarction, ANG II production and AT1 receptors are largely increased at the infarct site (29, 31). ANG II has been indicated to stimulate cardiac oxidative stress, which triggers inflammatory response (14, 21), and TGF- synthesis, which promotes cardiac fibrosis (35, 38). Chronic administration of the AT1 receptor antagonist losartan has been demonstrated to reduce infarct size and interstitial fibrosis and improve cardiac function and survival.

In this study, we adapted a series of novel techniques used to characterize tumor vascularity, perfusion, and levels of hypoxia (11, 20) to quantify the components and functionality (i.e., ability to deliver oxygen) of the vascular network at the infarct site at 1–4 wk after MI. We hypothesized that the dimensions and capacity of this vasculature to deliver oxygen in the infarct site are likely to be considerably less than that of the normal coronary circulation. These novel techniques were then used to examine the role for AT1 receptor binding in the structural remodeling of the intramyocardial coronary vasculature post-MI, hypothesizing that treatment with losartan can significantly improve vascular structure and function in MI rats. CD31 was used to visualize anatomical vessels and fluorescent staining with 3,3-diheptyloxycarbocyanine [DiOC₇(3)] was used to visualize patent vessels with blood flow (11) in the infarct site after permanent left coronary artery ligation. Immunohistochemical staining with 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5)/N,N'-(dipropyl)-tetramethyl-indocarbocyanine (Cy3) was used to quantify tissue hypoxia (11, 20). A combination of fluorescent and immunohistological stains was used to define the distribution of distances from cells to the nearest anatomical or perfused vessel.

	MATERIALS AND METHODS

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Model of MI. Six-week-old male Sprague-Dawley rats, weighing 250 g, were used. In brief, after induction of anesthesia with isoflurane, an anterior transmural MI was created by ligation of the left coronary artery with silk ligature as we have previously reported (32, 34, 35). MI was detected by evidence of an elevated S-T segment and the appearance of Q wave on an electrocardiogram, grossly visible scarring of the left ventricular free wall, and microscopic evidence of lost necrotic cardiomyocyte (32). Sham-operated, age/gender-matched rats served as controls. Sham operation involved an identical procedure, except the suture was passed around the vessel without ligation. All protocols were approved by the Animal Care and Use Committee of the University of Tennessee Health Science Center and followed the guidelines of the National Institutes of Health.

Losartan administration. Before surgery, treated animals received 15 mg/kg losartan by gastric gavage. During the 2-wk recovery period, treated animals were given 15 mg·kg^–1·day^–1 (2 times/day) losartan by gastric gavage. We did not observe a significant change in the blood pressure of MI rats treated with this dose of losartan (data not shown).

Experimental protocol. Hearts were removed at 1–4 wk post-MI (5 animals/time point). Rats were sedated, and EF5 (National Cancer Institute) was injected intraperitoneally and intravenously. Later (6 h), DiOC₇(3) (Molecular Probes) was injected via tail vein; 1 min later, animals were killed, hearts were removed, and ventricles were gravity filled with OCT to prevent chamber deformation, immediately quick-frozen using dry ice (total time <1 min), and stored at –80°C. Hearts were sectioned (10 µm thickness) at –20°C using a cryostat. Sections were mounted on poly-L-lysine-coated glass slides for later staining and imaging.

Visualization of anatomical and perfused vessels and levels of hypoxia. Location of all coronary vessels, referred to as anatomical vessels, was determined using CD31 (platelet endothelial cell adhesion molecule-1; BD Biosciences)/3-amino-9-ethylcarbazole (AEC; DAKO) staining (11). To distinguish perfused from anatomical vessels, fluorescent dye DiOC₇(3) was injected intravenously (1.0 mg/kg in 75% DMSO) 1 min before the heart was removed and rapidly frozen. This dose of DiOC₇(3) has been shown to provide optimal visualization of perfused vasculature by preferentially staining cells immediately adjacent to perfused vessels (10, 11). Following the method of Fenton et al. (11), EF5, a pentafluorinated derivative of etanidazole, along with one of its highly specific antibodies, ELK3–51 (Department of Radiation Oncology, The University of Pennsylvania Health System), were used to quantify levels of tissue hypoxia (16, 20, 22). This technique involves the oxygen-dependent metabolic activation of a nitroheterocyclic drug, leading to adducts (EF5 reduction-protein conjugates) formation between EF5 and cellular macromolecules. These adducts are formed at a much greater rate in hypoxic than aerobic cells (20). EF5 (3 ml of 10 mM EF5) was injected by tail vein. Later (15 min), another 3 ml EF5 (10 mM) were injected intraperitoneally (22), and 6 h later hearts were removed and frozen. Regions of high EF5 metabolism in myocardium were visualized immunochemically using the fluorochrome Cy3 conjugated to the ELK3–51 antibody. EF5 has been shown to reach well-perfused and poorly perfused areas of the tissue by diffusion from the microcirculation (10). The intensity of EF5/Cy3 has been shown to be inversely proportional to the degree of tissue hypoxia, changing by two orders of magnitude between 0.1 to 10.0% oxygen with constant values above and below this range (15).

On day 1 of analysis, slides were imaged for DiOC₇(3) to map the location of perfused vessels. After DiOC₇(3) imaging, slides were fixed in cold (–10°C) acetone for 5 min in a Coplin jar then air-dried for 5 min. Slides were then washed three times in Ca²⁺-free PBS and 5% normal rat serum (Jackson Immunoresearch) in buffer at room temperature. Slides were then washed three times in PBS, one time in Dako Peroxidase Blocking Reagent at room temperature, and three more washes in PBS. Finally, slides were incubated with 100 µl mouse anti-rat CD31 antibody overnight (1:100 dilution in Dako's Background Reducing Agent; Pharmingen) at 4°C.

On day 2 of analysis, after slides were thawed for 30 min at room temperature, they were washed three times with PBS at room temperature and were then incubated with 100 µl horseradish peroxidase-rat anti-mouse IgG (Jackson Immunoresearch) at 1:25 dilution in Dako's Background Reducing Agent (40 µg/ml) for 1 h at room temperature. Slides were then washed three times with PBS, incubated with 100 µl AEC substrate for 30 min at room temperature, and washed with PBS and double distilled water. They were then incubated with 200 µl of 5% normal rat serum in buffer for 30 min at room temperature, washed with PBS, and incubated with 100 µl ELK3.51-Cy3 (75 µg/ml) overnight at 4°C. Tissue sections treated with the secondary, but not the primary, antibody were used as negative control. On day 3, slides were washed with PBS + polyoxyethylenesorbitan monolaurate (Tween 20; Sigma) and PBS. The imaging system was calibrated with Cy3 calibration dye.

Imaging equipment and procedures. Stained sections were imaged using an epifluorescence inverted TE200 Nikon microscope equipped with a motorized stage (model no. 99S008; LEP) controlled by a computer using ImagePro (Media Cybernetics). This computerized microscope system was used to obtain matching images for the same tissue after several different staining procedures. Each section was scanned under three different staining conditions as follows: 1) epi-illumination images of the fluorescent DiOC₇(3) staining were obtained immediately after 10-µm sections were prepared; 2) on day 3, after the completion of immunohistochemical staining procedures, the same heart section used for DiOC₇(3) imaging was rescanned under epi-illumination, and matching fluorescent red-orange montages were obtained showing the distribution of EF5/Cy3 staining, which are indicative of hypoxia; and 3) with the use of transmitted light (100 W halogen), matching brownish-red montages of the CD31 staining were acquired.

In this system, the x-y spatial coordinates for imaging DiOC₇(3)-stained vessels were recorded automatically on a computer. After the first scan, the x-y spatial coordinates recorded were used to return to these specific positions (with a spatial repeatability of <2 µm) to take the matched EF5/Cy3 and CD31/AEC images. The intensity values of EF5 images were recorded in each field to quantify the degree of hypoxia in regions of the tissue corresponding to those obtained for CD31 and DiOC₇(3) images. These images were then superimposed over the corresponding CD31- and DiOC₇(3)-stained images to quantify the relationship between hypoxia and anatomical and perfused vascular configurations, respectively.

All fluorescent images of the EF5 marker of hypoxia were obtained before the use of transmitted light for imaging of CD31 staining to prevent fading of the EF5 staining. The number of vessels and their size distribution were quantified in the infarct site, excluding the necrotic band, for statistical comparison with controls by a blinded observer. CD31 images were enhanced using ImagePro to identify AEC-stained blood vessels. Similarly, DiOC₇(3)-stained images were enhanced to determine the location of perfused vessels. Distribution of diffusion distances was determined as the measured distance from every point in the tissue to the nearest perfused blood vessel, and are represented by a cumulative frequency of "distance to the nearest perfused vessel."

Statistical analysis. One-way ANOVA with planned contrasts was used to determine significant differences among experimental groups. Kolmogorov-Smirnov Test (StatGraphics Plus; Manugistics) was used to compare frequency distributions. All data are reported as means ± SE. All values of P < 0.05 were considered statistically significant.

	RESULTS

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Rat hearts from sham-operated controls and 1–4 wk post-MI were processed to quantify the density of anatomical and perfused vessels and levels of hypoxia. Necrotic tissue in the center of infarct was identified by a paucity of vasculature in general and perfused vessels in particular and by a relative lack of EF5/Cy3 staining. This necrotic tissue was not vascularized during 1–2 wk post-MI but was somewhat vascularized by 4 wk post-MI. The number of anatomical and perfused vessels and levels of hypoxia were not significantly different between the noninfarct site of infarcted hearts and normal tissue from sham-operated animals (data not shown).

As an example, Fig. 1A shows the colocalization of DiOC₇(3) and EF5/Cy3 images at 3 wk post-MI at low magnification. Red staining (EF5/Cy3) identifies hypoxic areas, whereas green staining [DiOC₇(3)] marks perfused vessels. The higher the intensity of the red stain, the higher the level of hypoxia. Figure 1 shows high-magnification images of normal tissue (B) and the infarct site (C) of the tissue section presented in Fig. 1A. As shown in Fig. 1, there is an inverse relationship between the intensity of EF5/Cy3 staining and the number of perfused vessels such that, at the infarct site, the number of perfused vessels decreases after MI, whereas the level of tissue hypoxia (as indicated by the intensity of EF5/Cy3 stain; red intensity) increases.

View larger version (47K): [in this window] [in a new window]   Fig. 1. A: example of colocalization of 3,3-diheptyloxycarbocyanine [DiOC7(3)] and 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5)/N,N'-(dipropyl)-tetramethyl-indocarbocyanine (Cy3) staining in a coronal section of infarcted rat heart 3 wk after myocardial infarction (MI). Red staining (EF5/Cy3) marks hypoxic areas while green staining [DiOC7(3)] marks perfused vessels. The infarct site is marked by arrows, and the necrotic band in the infarct site is marked by "N". The necrotic band is characterized by a lack of perfused vessels and a background level of EF5 staining. The higher the intensity of the red stain, the higher the level of hypoxia. High-magnification images of areas in the white boxes are shown in B and C. B: high-magnification image of normal region. C: high-magnification image of the infarct region. As the number of perfused vessels decreases after MI, the level of hypoxia (as indicated by the intensity of EF5/Cy3 stain; red intensity) increases.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Total number of all vessels (anatomical) and number of perfused vessels decreased significantly post-MI. In addition, the fraction of vessels that are perfused was reduced from 70% in normal to 40% in infarcted tissue (*P < 0.05 and **P < 0.01 compared with normal).

View larger version (19K): [in this window] [in a new window]   Fig. 3. Cumulative frequency of distance in the tissue to the nearest perfused vessel for various time points post-MI. Consistent with a decrease in the number of perfused vessels seen in Fig. 2, there was a significant (P < 0.05) and progressive increase in the distance to the nearest perfused vessel at weeks 1–4 post-MI.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Vessels in the 4-wk post-MI infarct area have significantly larger diameters compared with the normal tissue (*P < 0.05 compared with normal). Total vascular surface area post-MI was significantly less than normal (**P < 0.01 compared with normal) and did not change from 1 to 4 wk post-MI.

View larger version (11K): [in this window] [in a new window]   Fig. 5. Consistent with an observed decrease in the number of perfused vessels (Fig. 2) and an increase in the distance to the nearest perfused vessel (Fig. 3), a progressive increase in tissue hypoxia, as estimated from an increase in intensity of EF5/Cy3, was observed (**P < 0.01 post-MI compared with normal).

View larger version (33K): [in this window] [in a new window]   Fig. 6. Example of matching images of anatomical vessels (A), perfused vessels (B), and hypoxia levels (C) in the infarct site in losartan-treated animals 2 wk post-MI. The necrotic band in the infarct area, characterized by a lack of perfused vessels and background level of EF5 staining, is marked by "N."

View larger version (11K): [in this window] [in a new window]   Fig. 7. A: in losartan-treated animals, total number of all vessels and number of perfused vessels increased significantly post-MI. In addition, the fraction of vessels that are perfused was increased from 40% in untreated infarct tissue to 59% in the losartan-treated animals (*P < 0.05 compared with untreated animals 2 wk post-MI). B: in losartan-treated animals, the observed decrease in distance to the nearest perfused vessel (Fig. 3) and the increase in the number and fraction of perfused vessels (A) resulted in a significant decrease in tissue hypoxia (*P < 0.05 compared with untreated animals 2 wk post-MI).

	DISCUSSION

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To our knowledge, no other methodology for simultaneous measurements of vascular density, perfusion, and tissue hypoxia is currently available. Nevertheless, our measured vascular density in normal and MI hearts is in general agreement with those reported using more traditional techniques (1, 7, 28). Our results indicate that the number of anatomical and perfused vessels, and levels of tissue hypoxia, are not significantly different between noninfarct site of infarcted hearts and normal tissues from sham-operated animals. Cardiac hypertrophy may be characterized by abnormalities in myocardial perfusion (36), but the level and existence of hypertrophy may be dependent on the size and location of the infarct (2, 5, 26), and regional tissue perfusion in various parts of the heart is not significantly different between sham and MI animals (except for the infarct site of course; see Ref. 13). We did observe a small but significant increase in vessel density, but not perfusion, in the noninfarct site of losartan-treated animals, indicating that the ANG II pathway is important in vascular remodeling in both at and remote to the infarcted tissue. The observed increase in the fraction of perfused vessels at the infarct site in losartan-treated animals may be due to the fact that losartan inhibits the binding of ANG II at the AT1 receptor, which may cause vasodilation (27), resulting in a larger number of perfused vessels.

With the use of radiolabeled microspheres, a decrease in perfusion in the infarct site (40% of normal at 3 wk post-MI) has been reported (13). Using DiOC₇(3) fluorescent staining, we have shown a similar trend of decrease in the number of perfused vessels in the infarct site (23% of normal at 3 wk post-MI). It should be noted that injected microspheres provide an index of blood perfusion [similar to DiOC₇(3) staining] but not oxygen availability and/or hypoxia (as provided by EF5/Cy3 staining). Our hypoxia measurements, consistent with the DiOC₇(3) perfusion measurements, indicate that there is a >100% increase in our EF5/Cy3 intensity in the infarct site. This direct index of tissue hypoxia is not available with traditional measures such as vessel perfusion, and our EF5 measurements are consistent with a recent study demonstrating increased HIF-1 upregulation in human hearts from patients with ischemic cardiomyopathy (24).

Neovasculature begins to form in the infarct site within a few days after the infarction (4), but the high levels of hypoxia observed in our studies suggest that this neovasculature may not be adequate to accommodate the oxygen demands of myofibroblasts and may even be less likely to have the capacity to deliver (conductance vessels) and exchange (microvessels) the oxygen needed by functioning cardiomyocytes. There may be a short window of opportunity for in situ reconstitution of myocardial tissue, since fibrillar collagen becomes evident on day 7 and progressively replaces infarcted myocardium with a densely packed fibrillar scar by day 21 (3, 29). Because of the difficulties associated with growing new vessels in a densely packed fibrillar scar, the optimal time to regrow myocardium and its supporting vascular network may therefore be confined to the first 14 days post-MI. In this study, losartan treatment during the first 2 wk post-MI significantly moderated the observed reductions in vascular capacity and function.

In vivo, both angiogenesis and vasculogenesis can lead to the formation of new endothelial (and other vascular) cells. Rebuilding lost myocardial tissue, using such approaches as stem cell therapy (8), must include a vascular network able to nourish new cardiomyocytes under diverse physiological conditions. Many proangiogenic and provasculogenic compounds, including losartan, have undesirable side effects when administered systemically. We are currently developing a methodology to selectively deliver proangiogenic and provasculogenic compounds to the infarct site during this time frame (39).

In this study, we have successfully developed a series of novel techniques to quantitatively measure vascular composition and function and applied them to explore the role of angiotensin in cardiac vascular remodeling post-MI. This methodology is especially useful because various quantitative measures of tissue oxygenation (e.g., vascularity, perfusion, and tissue hypoxia) can be obtained in the same tissue section. Our measurements of vessel diameters are limited by the fact that they are indirectly estimated from CD31/AEC staining of the endothelium. Absolute values of diameters obtained from AEC or DiOC₇(3) staining may be different from the actual lumen diameter of in vivo perfused vessels. However, our approach provides a reliable estimate of relative changes in vessel diameter under different conditions (9).

	GRANTS

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M. F. Kiani is an Established Investigator of the American Heart Association. This work was supported by National Science Foundation Grant BES0090009 (M. F. Kiani), National Heart, Lung, and Blood Institute Grants HL-67888 (Y. Sun) and HL-62229 (K. T. Weber), and the Center of Excellence for Diseases of Connective Tissue at the University of Tennessee Health Science Center.

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. F. Kiani, Dept. of Mechanical Engineering, 1947 North 12th St., Philadelphia, PA 19122 (E-mail: mkiani{at}temple.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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