Fibroblast growth factor 2 control of vascular tone

Abstract

Vascular tone control is essential in blood pressure regulation, shock, ischemia-reperfusion, inflammation, vessel injury/repair, wound healing, temperature regulation, digestion, exercise physiology, and metabolism. Here we show that a well-known growth factor, FGF2, long thought to be involved in many developmental and homeostatic processes, including growth of the tissue layers of vessel walls, functions in vascular tone control. Fgf2 knockout mice are morphologically normal and display decreased vascular smooth muscle contractility, low blood pressure and thrombocytosis. Following intra-arterial mechanical injury, FGF2-deficient vessels undergo a normal hyperplastic response. These results force us to reconsider the function of FGF2 in vascular development and homeostasis in terms of vascular tone control.

Fig. 1

Generation of FGF2-deficient mice, a, Targeting scheme for generation of an Fgf2 null allele in mouse. The upper line (wild-type allele) represents a 7.0-kb Scrcl/Hindlll fragment of the wild-type Fgf2 genomic DNA, which contains the promoter region and the first exon (filled box). The targeting construct contains a 0.7-kb short homology arm at the 5′ end and a 3.0-kb long homology arm at the 3′ end of the Hprt minigene (open box). A thymidine kinase gene (hatched box) is used for negative selection. Homologous recombination between the wild-type allele and the targeting construct deletes part of the promoter and entire first exon of the Fgf2 gene (targeted allele). Long arrows represent the PCR primers used to identify the targeted allele; arrowheads represent primers used to identify the wild-type allele. Probe A is a 0.6-kb “outside” probe and probe B is a 0.9-kb “inside” probe. B: BamHI, H: Hindlll, N: Narl, S: Sacl, X: Xbal. b, Southern blot analysis of DNA from electroporated ES clones following HAT and gancyclovir selection. A representative clone, G8, is shown. DNA was digested with different restriction enzymes and probed with either probe A or probe B. The expected bands for BamHi digestion: wild-type (WT), 5.0 kb; mutant (Mut), 7.5 kb. For Socl digestion: WT, 4.2 kb, Mut, 6.7 kb. For H/ndlll digestion: WT, 4.2 kb, Mut, 3.9 kb. c, Southern blot analysis of viable offspring born from parents heterozygous for the targeted allele. DNA isolated from tail clips was digested with Hindlll and probed with probe B. Wild-type mice (+/+) show a 4.2-kb band and mutant mice (−/−) show a 3.9-kb band. Heterozygous mice (+/−) show both the 4.2- and 3.9-kb diagnostic bands, d, Northern blot analysis of RNA isolated from E1 3.5 embryos and several adult tissues from 2-month-old mice from heterozygous matings. Probing poly(A)+ RNA (5 μg) message with a probe consisting of exon 2 and 3 of Fgf2 detects a 5.9-kb Fgf2 message in tissues from wild-type (+/+) mice but not in knockouts (−/−) even after extended exposure for 104 h (not shown). With mouse Fgf1 cDNA as probe, a 3.4-kb and a 2.1-kb mRNA are detected in adult brain, heart and kidney. With mouse Fgf5 cDNA as probe, Fgf5 is very abundantly detected (predominantly as a 2.4-kb species) in skin. The band immediately below the 2.4-kb Fgf5 mRNA is the residual β-actin signal after stripping. Hybridization with a β-actin probe served as RNA loading control. Note that expression of Fgf1 and 5 was similar in Fgf2^+/+ and Fgf2^−/− mice, e Western blot analysis of FGF2 protein in brain. Brain from Fgf2^+/+ and Fgf2^+/− mice expressed an 18-kDa FGF2 as well as two high molecular mass species of 21 and 22 kDa. All three protein species were absent from Fgf2^+/+ mice.

Fig. 2

Contractile activity of isolated portal veins, a, Representative tracing of a pair of portal veins showing isometric force vs time for the spontaneous myogenic contractions, b, Tension-time integrals for isolated portal veins stimulated by increasing concentrations of phenylephrine, a vasoconstrictor. Each data point represents the mean (± s.e.m. for five pair of mice) of the tension–time integral generated from each portal vein measured for 60 s at each concentration of phenylephrine.

Fig. 3

Vascular hyperplasia following carotid injury occurs in Fgf2^+/+ mice. Carotid arteries were injured in adult mice and examined for the development of hyperplastic vascular layers. a–d, Hematoxylin and eosin (H&E)-stained sections from vessels 1 day after injury from control (Fgf2^+/+, Fgf2^+/+ n = 4) and Fgf2^−/− (n = 5) mice show a range of injury features from (a) endothelial cell denudation with platelet deposition (open arrowheads) proximal to the heart (prox) to (b) severe medial damage leading to medial hemorrhage (arrow) distal to the heart near the thyroid (dist). c, H&E-stained section of an Fgf2^−/− mouse carotid showing severe medial injury, d, Serial section of c stained with an elastin/van Gieson stain (e/VG) to highlight the elastic layers, which remained intact (open arrows), e–p H&E- and e/VG-stained serial, section pairs from 4-week post-injury carotids from Fgf2^−/− (n = 3) and Fgf2^+/+ (n = 2) mice show hyperplastic responses involving the adventitial (*), medial (white arrow), and intimal (closed arrow heads) layers. Panels e, f, i, and j are from the same wild-type mouse showing lesions from two positions (dist and prox). Panels g, h, k, and l show lesions from the same Fgf2 knockout mouse. Panels m, n, o, and p show lesions at the middle position (mid) along the injured carotid from a wild-type (m, n) and Fgf2^−/− mouse (o, p). The scale bar shown in d applies to all 1-day panels; the dimension scale in p is the same for all 4-week panels.

Fig. 4

Increased expression of Fgf2 mRNA in injured carotid arteries. RTPCR of Fgf2 transcripts from wild-type injured and noninjured, bilateral control carotids at 2, 4 and 6 days after injury. Representative results from one of the two experiments performed at each time point are shown. After transcribing mRNA into cDNA, serial dilutions (as indicated) were used in the reaction to ensure linear amplification. Amplification of glyceraldehyde phosphodehydrogenase (GAPDH) or β-actin transcripts served to control for template quantity.