C O V E R S T O R Y

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n the University of Pittsburgh Departmentof Surgery, they are tracking a phantom sus-pect. This ghost has given former presiden-tial candidate Bob Dole a new career in TV

commercials. (It is the basis for Viagra, the much-hyped magic potion for erectile dysfunction.) Italso appears to play a key role in virtually everyaspect of everyday physiology, from control of

IS U R G E O N S T R A C K A P O W E R F U L

B U T E L U S I V E M O L E C U L E

B Y E D W I N K I E S T E R J R .

I L L U S T R A T I O N | M I K E M C Q U A I D E

P O R T R A I T P H O T O G R A P H Y | J I M J U D K I S

GONE

HERE,

THEN

A P R I L 17

18 P I T T M E D18 P I T T M E D

blood pressure to wound healing to microbialinfection, even to childbirth.

Therapeutic applications have saved lives inthe operating room and given support to thosewith breathing problems. It also may help dial-ysis and coronary-bypass patients.

Yet this phantom comes and goes before itcan readily be detected.

As molecules go, nitric oxide, or NO, isboth elusive and unique, which helped itremain terra incognita until recently. Mostsubstances enter cells via specific receptors onthe cell’s surface, as a key fits into a keyhole;nitric oxide needs no receptors. It does its workas a gas, readily passing through the permeablecell barriers. NO also has an extremely briefhalf-life, a mere six seconds. It materializes inquick puffs and is gone soon after.

The molecule has captured the imagina-tion of thousands of researchers. A torrent of30,000 (and counting) NO papers haspoured out of laboratories, mostly in the lastfive years; Science magazine crowned it“Molecule of the Year” in 1992 and anIndian journal upped that to “Molecule ofthe Millennium.”

Much of the basic biology of this ghostsubstance has been unveiled, surprisingly, atthe laboratory benches housed in a surgicaldepartment—Pitt’s, to be exact. Formerdepartment chair Richard L. Simmons pointsout that surgeons’ research interests are nor-mally pegged as short-lived. In a markeddeparture from that stereotype, the Pitt teamhas zeroed in on pure science as the best routeto identifying NO’s benefits to patients.

“It is unusual,” says Simmons, who gaveup the department chair in 1998 after 11years, “for a surgery department to maintainsuch a devoted and unremitting focus on anyproblem in basic science.”

That dedication by a small group of sur-

geons has produced a series of research firsts.To name only a few of the most important,Pitt surgeons were first to show that NOcould be made in human liver cells (previous-ly it had been identified only in mouseimmune-system cells); first to show that it wasmade also by the heart; first to describe its rolein facilitating organ transplant rejection; firstto show that it could be used for therapy inblood-vessel disorders. “And lots more,”Simmons says. Perhaps the two most atten-tion-getting discoveries came in the early ’90sfrom Timothy Billiar, the tall, reservedNebraskan who led the research effort at Pittand last fall succeeded Simmons as depart-ment chair, and David Geller, now the SamuelP. Harbison Assistant Professor of Surgery anda faculty member at the Thomas E. StarzlTransplantation Institute. Billiar et al. identi-fied the human gene that turns on induciblenitric oxide synthase (iNOS), which is a keyenzyme for triggering production of NOthroughout the body. Geller cloned the gene.That success opened the way for expression ofa recombinant human iNOS. (Recombinant,meaning Geller applied the same gene-splic-ing technology—invented, in part, by Pittgrad Herb Boyer—that made possible treat-ments such as the now widely used form ofinsulin that has replaced animal-derivedinsulin. The other often caused allergic reac-tions in diabetics.) Pitt now holds four patentsbased on the cloned gene, and pharmaceuticalcompanies worldwide are assiduously usingthem to pursue a wide range of therapeuticapplications.

Oddly, although it combines the two mostcommon elements in the earth’s atmosphere—nitrogen and oxygen—little was understoodabout nitric oxide as recently as 15 years ago.What was known cast NO as an environmen-tal villain, a particularly noxious product of

auto exhaust. In the days before catalytic con-verters were mandatory, for example, Germanforesters and Greens vociferously blamed auto-generated NO for killing the spruces and firs ofthe beloved Black Forest.

And yet physicians had been using nitricoxide therapeutically for a century withoutcompletely understanding why. That was inthe form of nitroglycerin, a vasodilator oftenprescribed for angina pectoris (the acutechest pain that comes from heart musclesnot receiving enough blood) and other car-diac conditions. Nitroglycerin expands thediameter of blood vessels and increases bloodflow to the heart.

In 1977 Ferid Murad, a pharmacologist/physiologist at the University of Texas–Houston, discovered that nitroglycerinopened coronary arteries by releasing NO. Hetheorized that the body itself could stimulatethe release of NO to regulate blood vessels.But he had no experimental evidence to backup his theory. Then in 1980 Robert F.Furchgott of the State University of New YorkHealth Science Center at Brooklyn found thatthe endothelium, the inner lining of the bloodvessels, released a substance that relaxed thevascular muscles and increased blood flow. Hecalled the substance endothelium-derivedrelaxing factor, or EDRF.

It took six years more for Louis J. Ignarroof the University of California, Los Angeles,another pharmacologist, to capture the ghost.Ignarro, using spectroscopy, identified therelaxing factor as nitric oxide. Furchgott,Murad, and Ignarro received the 1998 NobelPrize in Physiology or Medicine for discover-ing “an entirely new principle for signaling inthe human body.”

In 1987, as Furchgott and Ignarro caughtthe attention of the scientific world, RichardSimmons came to Pitt from the University of

Normal Injured Treated with NO

Science magazine once crowned

nitric oxide the “Molecule of the

Year.” Much of what we know

about it comes from Pitt’s sur-

gery department. On the left, an

uninjured arterial wall from a

rat. In the middle frame, arterial

injury resulted in the overgrowth

of smooth muscle cells with

narrowing of the artery. On the

right, treatment of the injured

artery with nitric oxide gene

therapy inhibited this overgrowth

and preserved the normal archi-

tecture of the arterial wall.

Minnesota. Billiar, four years out of medicalschool, came with him as a research fellow.Simmons, “a transplanter by trade,” quicklysaw that nitric oxide, with its ability to dilateblood vessels, might offer clues to one of themost dismaying and unfortunately all toofamiliar problems of the operating room—the postsurgical shock and loss of blood pres-sure that can lead to massive organ failure anddeath. Geller, who joined the effort a fewyears later, had more than once seen these

devastating effects: “Patients get sick and thenvery sick and end up in surgical IC units; theydevelop sepsis or disseminated infectionthroughout the entire body, and go into mul-tisystem organ failure, which eventually doesthem in; they succumb and die.”

Simmons explains how the researchoffensive came about. “I am a catalyst bynature—and curious.” He began to recruit ateam to carry out the work.

“The power structure in the field of sur-gery is such that a chairman has tremendouspower,” he says. “Therefore, people in thefield with ambition tend to be drawn to thechair’s interests. A smart chair takes advan-tage of that opportunity. You get to choosethe smart ones. You support the careers of thesmartest and most productive and are reward-ed for that in turn.”

By 1988, Simmons was starting to see thereturns on his recruiting efforts in the form

of research results; by 1993 he was floodedwith NO breakthroughs. “We didn’t at firstfocus on NO,” Simmons says. “It just hap-pened to be the answer to questions we wereasking about organ failure in sepsis. Thatcaused us to ask more questions. If you keepasking why, you sometimes are lucky.”

Nitric oxide actually plays a couple ofbasic roles in human biology, each of whichis controlled by the expression of different

genes. When it’s released by an enzymeknown as constitutive nitric oxide synthase(cNOS), it often becomes a neurotransmit-ter, one of the chemical messengers that car-ries impulses from nerve cell to nerve cell.That enzyme is present in minute quantitiesin the body at all times to carry out this role.Other times, it’s turned on by induciblenitric oxide synthase (iNOS)—the enzymeBilliar and Geller have come to know aboutas well as anyone—and it is produced whenthe body is stressed by inflammation, say, orwidespread infection. Then it is turned outin great quantity—up to 1,000 times morethan when it’s produced by cNOS. This lat-ter, often crisis, mode of nitric oxide is whathas captivated Billiar and Geller.

Billiar led the way in unraveling the com-plexities of the molecule. As early as 1989,he was explaining that NO was derived fromL-arginine, one of the amino acids that arethe building blocks of life. He also identifiedthe role of growth-regulating cytokines inthe generation of NO. His curriculum vitaelists 24 pages of publications, almost all onNO, and nine pages of invited lectures

Timothy Billiar led the way in unraveling

the complexities of the molecule. His CV

includes some 24 pages listing his publi-

cations on nitric oxide.

A P R I L 19

This ghost has given former presidential candidate

Bob Dole a new career in TV commercials.

20 P I T T M E D

click it again and then pointing to the slen-der wires. “I’m a liver surgeon. I spend halfmy time doing liver transplants, half remov-ing liver cancers. With this, we click it intoa liver cancer and these 10 little tines, orhooks, come out and burn out the cancer.

“If we can’t cut ’em out, we burn ’em out.” Geller is certainly a preeminent researcher

as well as surgeon. He has 60 NO publica-tions on his own CV, holds a career develop-ment award from the American College ofSurgeons and a five-year $500,000 researchgrant from the National Institutes of Health(NIH). After conducting liver research atNIH, he came to Pitt as a 25-year-old intern,quickly immersing himself in the nitric oxidework. His iNOS cloning triumph camebefore he was 30. Billiar calls him “the world’sleading authority on the iNOS gene.”

Geller continually emphasizes the depart-ment’s view that, despite its commitment tobasic science, Pitt surgeons remain surgeonsfirst and foremost.

“My research interests stem from what Ido clinically as a surgeon,” he says.

“Many problems we see in the operatingroom, if we could understand the pathophys-iology or molecular biology and the basicmechanisms responsible, maybe ultimatelywe could find a way to prevent or find novelstrategies to treat patients.”

Thus, the vexing problem of postsurgicalshock remains at the top of the agenda for thedozen or so department “worker bees,” asSimmons calls the NO researchers. Billiar isthe primary investigator (working with BrucePitt of environmental and occupational health,Tony Bauer of medicine, Brian Harbecht ofsurgery, Mitch Fink of critical care medicine,and others) on a major, nationally fundedeffort that’s looking into this complex prob-lem. Department researchers are also investi-gating NO’s apparent role in apoptosis, or pro-grammed cell death. “We are one of many labsall over the world looking into this question,”Billiar says, noting that understanding NO’slink is hugely important. “It’s a big field ofresearch,” he notes. For instance, researchersthink that understanding NO’s role in cell-death programming might lead to new cancer

around the world. Within seven years herose from assistant professor of surgery toassociate professor to Watson Professor ofSurgery to George Vance Foster Professorand chair of the department.

“Tim is recognized as one of the world’sleaders in this area,” Simmons says, praisinghis protégé. “He is always invited to meet-ings, national and international, on anythingto do with NO—a testimony to the impor-tance of his work.”

In contrast to Billiar, who is often so soft-spoken that you must lean forward to catchwhat he is saying, the ebullient Geller comesbounding from his office, hand outstretched.“I’m Dave Geller!” he says, without waitingfor you to be ushered in by an assistant. He sitsat a cluttered desk and scoops up a pencil-sizedapparatus, clicks it, and hands it to his visitor.Tiny wire hooks pop out at the base.

“For radiofrequency ablation of livertumors,” he explains, inviting the visitor to

After Timothy Billiar found the gene,

David Geller (above) cloned it.

therapies down the road—however, no oneknows how NO might halt cancer growth, itmay actually stimulate it.

NO zaps infections as well; elsewhere atPitt, researchers are studying how theimmune system employs it in diseases suchas tuberculosis (no one understands exactlyhow the TB process works). Also underinvestigation is NO’s possible role in neu-rodegenerative diseases such as Parkinson’sand Alzheimer’s as well as rheumatoid arthri-tis and osteoarthritis. Too much NO pro-duction triggered by inflammation may havea role in tissue destruction in arthritic joints.

On a more basic level, Geller is investigat-

ing, through an NIH grant, the on/off signalsof the iNOS gene. “We have a fairly good ideaof what turns on the gene. Almost nothing isknown about the molecular mechanisms thatturn off the gene. In certain settings youwant to overexpress the gene because it would be beneficial; in other settings itwould be clearly harmful, and you want toturn it off or prevent its being activated.” Togive an example of an NO too-much/too-little tightrope walk that occurs naturally inour bodies: The cardiovascular system relieson a little NO to keep blood pressure incheck, yet an excess can bring about cata-strophic collapse.

A clinical trial of NO in kidney dialysispatients is in the startup mode, directed byvascular surgeon Edith Tzeng, assistant profes-sor of surgery (who, Billiar says, in his low-keyway, “happens to be my wife”). In dialysis, aU-shaped arteriovenous shunt linking arteries

Nitric oxide has an extremely brief half-life, a mere

six seconds. It materializes in quick puffs and is gone

soon after.

As part of a huge gene therapy grant, Edith

Tzeng is infusing the iNOS gene to see if it will

stop blood vessel walls from narrowing. By the

way, she determined that nitric oxide can ham-

per growth of smooth muscle on vessel walls.

and veins is permanently implanted, usually inthe arm, and blood is withdrawn via the vein,cleansed of water and impurities and thenreturned via the artery. The shunts often nar-row and allow clots to develop, or they closeup altogether and collapse within a year or soafter implantation. In earlier research, Tzeng,with Pitt’s Larry Shears, had shown that NOinhibited the growth of smooth muscle in thevascular walls; it is this growth that reducesthe diameter of the blood vessel and hampersblood exchange. In the trials, supported aspart of a $14 million cardiac gene therapygrant, she will infuse the cloned iNOS genewhen the shunt is implanted to determine ifthe vessels will remain open. Later, the inves-tigation may be extended to coronary-bypassand angioplasty patients, whose vessels alsomay close some years after the procedure.

Reflecting on the NO furor of the pastdecade, Simmons notes that worldwideresearch interest in the subject has started toplateau. There is no sign of a slowdown atPitt, however, where surgeons continue tochase the shadow. �

William Kiester contributed to this article.