Full Text HL-97-014
NIH Guide, Volume 26, Number 27, August 15, 1997
RFA: HL-97-014


National Heart, Lung and Blood Institute
Letter of Intent Receipt Date: January 5, 1998
Application Receipt Date: March 26, 1998
This solicitation invites research grants focused on how HIV
infection alters the expression of endothelial cell genes thereby
modifying the normal structure and function of the endothelium and
exposing organs to HIV-infected cells and to circulatory factors that
could cause damage.  Ultimately, the goal of this solicitation is to
contribute to knowledge that might lead to new approaches to prevent
HIV associated dysfunction and degeneration caused by HIV to vital
organs, including lungs, heart, bone marrow, and the vasculature.
The Public Health Service (PHS) is committed to achieving the health
promotion and disease prevention objectives of "Healthy People 2000,"
a PHS-led national activity for setting priority areas.  This Request
for Applications (RFA), "Endothelial Dysfunction in HIV Infection" is
related to the priority area of HIV Infection.  Potential applicants
may obtain a copy of "Healthy People 2000" (Full Report: Stock No.
017-001-00474-0) or "Healthy People 2000" (Summary Report:  Stock No.
017-001-00473-1) through the Superintendent of Documents, Government
Printing Office, Washington, DC 20402-9325 (telephone 202-512-1800).
Applications may be submitted by domestic, for-profit and non-profit
organizations, public and private, such as universities, colleges,
hospitals, laboratories, units of State and local governments, and
eligible agencies of the Federal government.  Racial/ethnic minority
individuals, women, and persons with disabilities are encouraged to
apply as Principal Investigators.  Applications from foreign
institutions will not be accepted.  However, subcontracts to foreign
institutions are allowable, with sufficient justification.
All current policies and requirements that govern the research grant
programs of the NIH will apply to grants awarded under the RFA.
Awards under this announcement to foreign institutions will be made
only for research of very unusual merit, need, and promise, and in
accordance with Public Health Service policy governing such awards.
Among the disciplines and expertise that may be appropriate for this
research program are cell biology, molecular biology, developmental
biology, biochemistry, genetics, immunology, pathology, virology,
physiology, cardiology, hematology, and pulmonology.
This RFA will use the NIH individual research project grant (R01)
mechanism of support.  Investigators without prior R29 or R01 support
are encouraged to apply for this RFA and to identify their status in
a cover letter.  However, specific application instructions have been
modified to reflect "MODULAR GRANT" and "JUST-IN-TIME" stream-lining
efforts being examined by the NIH.  The modular grant concept
establishes specific modules in which direct costs may be requested
as well as a maximum level for requested budgets. Only limited
budgetary information is required under this approach.  The just-in-
time concept allows applicants to submit certain information only
when there is a possibility for an award.  It is anticipated that
these changes will reduce the administrative burden for the
applicants, applicant institutions, reviewers and Institute staff.
For this RFA, funds must be requested in $25,000 direct cost modules
and a maximum of eight modules ($200,000 direct costs) per year may
be requested.  A feature of the modular grant concept is that no
escalation is provided for future years, and all anticipated expenses
for all years of the project must be included within the number of
modules being requested.  Only limited budget information will be
required and any budget adjustments made by the Initial Review Group
will be in modules of $25,000.  Instructions for completing the
Biographical Sketch have also been modified.  In addition, Other
Support information and the application Checklist page are not
required as part of the initial application.  If there is a
possibility for an award, necessary Budget, Other Support and
Checklist information will be requested by NHLBI staff following the
initial review.  The APPLICATION PROCEDURES section of the RFA
provides specific details of modifications to the standard PHS 398
application kit instructions.
Responsibility for the planning, direction, and execution of the
proposed project will be solely that of the applicant.
This RFA is one-time solicitation.  Future unsolicited competing
continuation applications will compete with all investigator-
initiated applications and be reviewed according to the customary
peer review procedures.  It is anticipated that support for this
program will begin in September, 1998.  Administrative adjustments in
project period and/or amount may be required at the time of the
It is anticipated that for fiscal year 1998, approximately $2.0
million total costs will be available for the first year of support
for this initiative.  Award of grants pursuant to this RFA is
contingent upon receipt of such funds for this purpose.  It is
anticipated that up to eight new grants will be awarded under this
program.  Applicants may request up to five years of support.  The
specific number to be funded will, however, depend on the merit and
scope of the applications received and on the availability of funds.
Direct costs will be awarded in modules of $25,000 less any overlap
or other necessary administrative adjustments.  Indirect costs will
be awarded based on the negotiated rates.
Current treatment of HIV-infected patients has greatly improved
prospects for survival due to introduction of drugs to inhibit
replication of the virus.  A question that remains to be answered is
whether reduction in viral burden will result in retardation of, and
possibly permit healing of, organ damage.  Such damage appears to be,
in part, the result of transmission of HIV-infected cells across the
endothelium, usually without actual infection of endothelial cells
themselves, although whether endothelial cells can be infected by HIV
is still open to debate.  Under normal circumstances, the endothelial
cell barrier plays a critical role in the protection of tissues, but
it is clear that in HIV-infected patients this barrier fails,
permitting exposure to infections and other sources of damage.
Understanding how infected cells and soluble factors present in
circulating blood can change gene expression in endothelial cells
could lead to new therapeutic options for the treatment of HIV-
induced organ damage.
The endothelium, which forms a single cell layer throughout the
vascular system, performs multiple functions that are often modified
by regional location.  In addition to providing a balance between
vasoconstriction and vasodilation and anti- and pro-coagulation, it
is the source of growth factors and cytokines.  It expresses adhesion
molecules and receptors for local and circulating factors. It also
serves to control the influx of macromolecules into the extravascular
space.  A multitude of circulating factors can activate the
endothelium and lead to altered gene expression, thus producing
changes in structure and function.
En face studies of human aortic endothelium at autopsy have revealed
distinct differences between controls and HIV-infected patients.
Morphologic changes included disturbances of the regular pattern of
the endothelial layer and the presence of smaller cells with rounded
nuclei and increased chromatin as well as bizarre-shaped
multinucleated cells.  Increased adherence of leukocytes, mostly of
the monocyte/macrophage lineage, were observed in the endothelium.
Endothelial cells from the HIV-infected patients were found to
express increased amounts of some adhesion molecules and growth
factors and major histocompatibility complex class II antigen
(VCAM-1, ELAM-1, HLA-DR antigen, and IL-1beta).  On the other hand,
in vitro studies have shown that interleukin 2 (IL-2) secretion,
which is important for stimulation of T-cell responses, is diminished
in HIV-exposed endothelium.  How HIV infection causes these changes
in endothelial cells and alters their normal immunologic function
requires elucidation.
HIV-infected monocytes have been shown to exhibit increased adherence
to endothelial cells and to traverse the endothelial barrier.  One
study showed that monocytes accomplished this by producing
extracellular matrix-degrading metalloproteinases.  Interestingly,
tissue inhibitors of metalloproteinases, also produced by monocytes,
were increased after HIV infection but not in sufficient quantities
to inhibit degradation, suggesting that the balance of these two
classes of enzymes is perturbed by HIV.  Another study reported that
IL-1Beta-activated endothelium is disrupted by elastase secreted by
polymorphonuclear cells.  Yet another study, focussed on the
migration of lymphocytes through monocyte-conditioned endothelium,
showed no evidence of disruption of the endothelium.  In culture,
HIV-infected monocytes were shown not only to exhibit increased
adherence to microvascular endothelial cells (MVEC) from skin, brain
and lung but also to cause marked morphologic changes in MVEC.  These
changes included retraction from sites of contact at cell-cell
junctions without loss of cell viability as judged by trypan blue
exclusion.  HIV infection of monocytes increases their expression of
the cell adhesion molecules, LFA-1, LFA-2, and LFA-3.  Interaction
with endothelial cells involves receptors for these molecules as well
as adhesion molecules expressed on endothelial cells and their
receptors on monocytes.  TNFAlpha-activated MVEC showed increased
capacity for binding to HIV-infected monocytes and this may be of
importance in vivo where high serum levels of TNFAlpha and IL-1 have
been documented in HIV-infected patients.  TNFAlpha has also been
shown to be one of the cytokines produced by monocytes in response to
stimulation by HIV-1 glycoprotein 120.  The complexity of endothelial
cell-HIV relationships is further illustrated by the finding that
monocyte contact with endothelial cells upregulates HIV biosynthesis
in infected monocytes and may have significant impact on the
pathogenesis and tissue distribution of HIV infection.
Another source of endothelial damage is the tat protein, which is
known to be secreted extracellularly by infected circulating cells.
Human umbilical vein endothelial cells in culture have been shown to
respond to tat protein by upregulation of IL-6 production and the
expression of E-selectin adhesion molecules.  E-selectin is specific
for endothelial cells and is important for mediating binding of
polymorphonuclear cells, monocytes, natural killer cells and CD4+
memory T-cells to the endothelium.  IL-6 functions as a potent factor
for B-cell maturation and T-cell activation. It also acts on
endothelial cells to increase their permeability.  The mechanism by
which tat protein has these effects on IL-6 and E-selectin production
is unclear, although it has been shown to enter cells, to locate to
the nucleus and transactivate the HIV long term repeat (LTR). Three
mechanisms for tat activation of endothelial cells have been
postulated:  1) direct action to enhance transcriptional gene
expression of cell factors; 2) indirect enhancement of gene
expression by interactions with cell proteins; and 3) initiation of
signal transduction by binding to cell membrane receptors.  Brain
microvascular endothelial cells have also been shown to permit the
migration of HIV-1-infected monocytes into the brain by a mechanism
that involves induction of adhesion molecules as a result of soluble
factors present in the blood.
In a recently published paper it was reported that the tat protein
contains sequences (amino acids 42-64) similar to sequences of
molecules with angiogenic properties, e.g., FGF, VEGF-A, hepatocyte
growth factor, and heparin-binding growth factor.  A peptide
containing this domain was able to induce in vivo angiogenesis that
could not be inhibited by metalloproteinase-2.  Further studies
showed that tat acts through activation of Flk-1/KDR receptor.
Flk/KDR is important in vascular development for endothelial cell
proliferation and migration but not differentiation.  Thus it seems
that tat has activated an embryonic gene not normally expressed in
mature cells.  This activity of the tat protein could account for the
aggressiveness of AIDS-associated Kaposi's sarcoma and for the
increased incidence of tumors in AIDS patients.
It is possible that the altered expression of other genes involved in
vascular development and maintenance may hold the key to the
morphologic and functional changes that contribute to HIV
pathogenesis.  For example, recent research has shown that the
receptor tyrosine kinase TIE is important in late angiogenesis during
development and plays a key role in the survival or proliferation of
some, but not all, mature endothelial cell populations.  Yet very
little research has been conducted along these lines.  Another
neglected area is that of the lymphatic endothelium and its
interactions with lymphocytes.
The possibility that other HIV viral proteins might have sequences
that are similar to physiologically active peptides or proteins has
not been fully explored.  In 1992, Parmentier et al. showed that
epitopes of HIV regulatory proteins, tat, nef, and rev were expressed
in normal human tissues from various sites.  They found, for example,
that anti-HIV-1 tat antibodies labeled blood vessels from uninfected
individuals and that anti-rev antibody stained high endothelial
venules.  The significance of these findings remains to be examined.
In addition to the effects of virus-encoded proteins, the pro-
inflammatory molecules released by monocytes must also be considered,
both with respect to HIV infection and opportunistic infections such
as CMV.  Similarly, HIV-injured endothelium may play a role in
facilitating entry of opportunistic infections into target tissues.
Pulmonary hypertension, with histologic lesions indistinguishable
from primary pulmonary hypertension, is a well recognized but rare
complication of HIV infection. When present, the pulmonary
hypertension contributes significantly to morbidity and mortality.
The pathogenesis of this HIV-associated pulmonary hypertension
remains unclear, but a direct role of HIV is unlikely, since HIV has
not been found in the vascular endothelial cells of these patients.
However, an indirect role, secondary to chronic inflammation and
immune activation that accompanies HIV infection has been suggested.
Speculations include the possibility that HIV infection triggers
endothelial cell proliferation in some unknown way, perhaps as a
result of stimulation by growth factors.  The finding that
antiretroviral therapy appears to have a beneficial effect on
echocardiographic measures of right heart pressure gradients in HIV-
infected patients with pulmonary hypertension is consistent with such
an indirect inflammatory etiology.
Dysfunction of pulmonary vascular endothelium appears to be important
in the pathophysiology of primary pulmonary hypertension and is the
probable source of increased platelet activation, release of 5-HT,
and thomboxane, as well as reduced the amounts of nitric oxide and
prostacyclin.  Investigation of the pulmonary vascular endothelium in
HIV-associated pulmonary hypertension is likely to clarify the role
of endothelial dysfunction in HIV disease, and also lead to a better
understanding of primary pulmonary hypertension.
Despite the fact that endothelial cells lack CD4 receptors that are
thought to be essential for infection with HIV, microvascular
endothelial cells from bone marrow have been shown to be capable of
infection with the virus.  In these cells, IL-1`-induced release of
IL-6 and G-CSF is significantly reduced, possibly implicating this
deficiency in the hematopoietic malfunction observed during progress
of HIV disease.  Other reports suggest that umbilical vein
endothelial cells may be capable of infection with HIV. These
findings illustrate the variable properties of endothelial cells from
various tissues and emphasize the need for considering the diversity
of endothelial cells in seeking to elucidate the complexities of
endothelial-HIV interactions.
The emphasis of this solicitation is on how the sequelae of HIV
infection alter the expression of endothelial cell genes.  The
following topics are suggested as examples only. Applicants are urged
to consider other topics within the scope of this solicitation based
on their own knowledge of the field and their expertise.
o Investigation of mechanisms underlying altered gene expression of
adhesion molecules in endothelial cells exposed to soluble
circulating factors in HIV infection.
o Determination of local interactions between tissues and endothelial
cells as determinants of endothelial characteristics and
susceptibility to injury from HIV.
o Elucidation of the signaling mechanisms between monocytes and
endothelial cells that enhance HIV replication.
o Exploration of the potential for HIV proteins to interact with
cell-surface receptors to modify morphologic features and functional
activity of endothelial cells.
o Investigation of mechanisms underlying the loss of immunocompetence
of endothelial cells in HIV infection.
o Investigation of the role of the pulmonary vascular endothelium in
the pathogenesis of HIV-associated pulmonary hypertension.
Upon initiation of the program, the NHLBI will sponsor periodic
meetings to encourage exchange of information among investigators who
participate in this program.  Travel funds for a one day meeting each
year, most likely to be held in Bethesda, Maryland should be included
in the modules. Applicants should also include a statement in their
applications indicating their willingness to participate in these
It is the policy of the NIH that women and members of minority groups
and their subpopulations must be included in all NIH supported
biomedical and behavioral research projects involving human subjects,
unless a clear and compelling rationale and justification is provided
that inclusion is inappropriate with respect to the health of the
subjects or the purpose of the research.  This new policy results
from the NIH Revitalization Act of 1993 (Section 492B of Public Law
103-43) and supersedes and strengthens the previous policies
(Concerning the Inclusion of Women in Study Populations, and
Concerning the Inclusion of Minorities in Study Populations), which
have been in effect since 1990.  The new policy contains some
provisions that are substantially different from the 1990 policies.
All investigators proposing research involving human subjects should
read the "NIH Guidelines For Inclusion of Women and Minorities as
Subjects in Clinical Research," which have been published in the
Federal Register of March 20, 1994 (FR 59 14508-14513) and reprinted
in the NIH Guide for Grants and Contracts, Volume 23, Number 11,
March 18, 1994.
Investigators also may obtain copies of the policy from the program
staff listed under INQUIRIES.  Program staff may also provide
additional relevant information concerning the policy.
Prospective applicants are asked to submit, by January 5, 1998, a
letter of intent that includes a descriptive title of the proposed
research, the name, address, and telephone number of the Principal
Investigator, the identities of other key personnel and participating
institutions, and the number and title of the RFA in response to
which the application may be submitted.  Although a letter of intent
is not required, is not binding, and does not enter into the review
of a subsequent application, the information that it contains allows
Institute staff to estimate the potential review workload and avoid
conflict of interest in the review.
The letter of intent is to be sent to:
Dr. C. James Scheirer
Chief, Review Branch
Division of Extramural Affairs
National Heart, Lung, and Blood Institute
6701 Rockledge Dr., Room 7220, MSC 7924
Bethhesda, MD 20892-7924
Telephone: (301) 435-0266
FAX: (301) 480-3541
Email: James_Scheirer@NIH.GOV
Sample budgets and justification page will be provided upon request
or following the submission of a letter of intent.
The total direct costs must be requested in accordance with the
program guidelines and the modifications made to the standard PHS 398
application instructions described below:
Do not complete Form Page 4 of the PHS 398 (rev 5/95). It is not
required nor will it be accepted at the time of application.
Do not complete the categorical budget tables on Form page 5 of the
PHS 398 (rev.  5/95).  Only the requested total direct costs line for
each year must be completed based on the number of $25,000 modules
being requested. Applicants may not request a change in the amount of
each module.  A maximum of eight modules ($200,000 direct costs) per
year may be requested and each applicant may request up to five years
of support for this RFA.  Direct cost budgets will remain constant
throughout the life of the project (i.e. the same number of modules
requested for all budget periods).  Any necessary escalation should
be considered when determining the number of modules to be requested.
However, in the event that the number of modules requested must
change in any future year due to the nature of the research proposed,
appropriate justification must be provided.  Total Direct Costs for
the Entire Proposed Project Period should be shown in the box
-Budget justifications should be provided under "Justifications" on
Form Page 5 of the PHS 398.
-List the names, role on the project and proposed percent effort for
all project personnel (salaried or unsalaried)and provide a narrative
justification for each person based on his/her role on the project.
-Identify all consultants by name and organizational affiliation and
describe the services to be performed.
-Provide a general narrative justification for individual categories
(equipment, supplies, etc.) required to complete the work proposed.
More detailed justifications should be provided for high cost items.
Any large one-time purchases, such as large equipment requests, must
be accommodated within these limits.
o CONSORTIUM/CONTRACTUAL COSTS - If collaborations or subcontracts
are involved that require transfer of funds from the grantee to other
institutions, it is necessary to establish formal subcontract
agreements with each collaborating institution.  A letter of intent
from each collaborating institution should be submitted with the
application.  Only the percentage of the consortium/contractual TOTAL
COSTS (direct and indirect) relative to the total DIRECT COSTS of the
overall project needs to be stated at this time.  The following
example should be used to indicate the percentage cost of the
consortium, "The consortium agreement represents 27% of overall
$175,000 direct costs requested in the first year.". A budget
justification for the consortium should be provided as described in
the "Budget Justification" section above (no Form Page 5 required for
the consortium).  Please indicate whether the consortium will be in
place for the entire project period and identify any future year
changes in the percentage relative to the parent grant.
     If there is a possibility for an award, the applicant
will be requested to identify actual direct and indirect
costs for all years of the consortium.  Please note that
total subcontract costs need not be calculated in $25,000
modules.  However, when subcontract funds are added to the
parent grant budget, the total direct cost amount must be
included in the number of $25,000 modules requested.
o BIOGRAPHICAL SKETCH - A biographical sketch is required
for all key personnel, following the modified instructions
below.  Do not exceed the two-page limit for each person.
     -Complete the educational block at the top of the form
     -List current position(s) and those previous positions
directly relevant to the application;
     -List selected peer-reviewed publications directly
relevant to the proposed project, with full citation;
     -The applicant has the option to provide information on
research projects completed and/or research grants
participated in during the last five years that are relevant
to the proposed project.
o OTHER SUPPORT - Do not complete the "Other Support" pages
(Form Page 7).  Selected other support information relevant
to the proposed research may be included in the Biographical
Sketch as indicated above.  Complete Other Support
information will be requested by NHLBI staff if there is a
possibility for an award.
o CHECKLIST - No "Checklist" page is required as part of the
initial application.  A completed Checklist will be
requested by NHLBI staff if there is a possibility for an
o The applicant should provide the name and phone number of
the individual to contact concerning fiscal and
administrative issues if additional information is necessary
following the initial review.
Submit a signed, typewritten original of the application and
three signed, photocopies, in one package to:
6701 ROCKLEDGE DRIVE, ROOM 1040 - MSC 7710
BETHESDA, MD  20892-7710
BETHESDA, MD 20817 (for express courier service)
At the time of submission, two additional copies of the
application must be sent to Dr. C. James Scheirer, at the
same address given above in the section on LETTER OF INTENT.
Applications must be received by March 26, 1998.  If an
application is received after that date, it will be returned
to the applicant without review.  The Division of Research
Grants (DRG) will not accept any application in response to
this RFA that is essentially the same as one currently
pending initial review, unless the applicant withdraws the
pending application.  The DRG will not accept any
application that is essentially the same as one already
reviewed.  This does not preclude the submission of
substantial revisions of applications already re- viewed,
but such applications must include an introduction
addressing the previous critique.
Applications that are complete and responsive to the RFA
will be evaluated for scientific and technical merit by an
appropriate peer-review group convened in accordance with
NIH peer-review procedures.  As part of the initial merit
review, all applications will receive a written critique and
undergo a process in which only those applications deemed to
have the highest scientific merit, generally the top half of
applications under review, will be discussed, assigned a
priority score, and receive a second level review by the
National Heart, Lung, and Blood Advisory Council.
The personnel category will be reviewed for appropriate
staffing based on the requested percent effort.  The direct
costs budget request will be reviewed for consistency with
the proposed methods and specific aims.  Any budgetary
adjustments recommended by the reviewers will be in $25,000
modules.  The duration of support will be reviewed to
determine if it is appropriate to ensure successful
completion of the requested scope of the project.  Other
review criteria will include:
o scientific, technical or medical significance and
originality of proposed research
o appropriateness and adequacy of the experimental approach
and methodology proposed to carry out the research
o qualifications and research experience of the Principal
Investigator and staff, particularly, but not exclusively,
in the area of the proposed research
o availability of the resources necessary to perform the
The initial review group will also examine the provisions
for the protection of human and animal subjects and the
safety of the research environment.
Applications will compete for available funds with all other
approved applications.  The following will be considered in
making funding decisions:  quality of the proposed project
as determined by peer-review, availability of funds, and
program priority.
Inquiries concerning this RFA are encouraged.  Potential
applicants should request a copy of the full RFA which will
include sample budget pages as previously stated.  The
opportunity to clarify any issues or questions from
potential applicants is welcome.  Direct inquiries regarding
programmatic issues to:
Dr. Lan-Hsiang Wang
Heart Research Program, DHVD
National Heart, Lung, and Blood Institute
Two Rockledge Center, Suite 9044
6701 Rockledge Drive, MSC 7940
Bethesda, MD  20892-7940
Phone:  301-435-0510
Fax:  301-480-1335
E-mail: LW72F@NIH.GOV
Dr. Hannah H. Peavy
Division of Lung Diseases
National Heart, Lung, and Blood Institute
Two Rockledge Center, Suite 10018
6701 Rockledge Drive, MSC 7952
Bethesda, MD  20892-7952
Phone:  301-435-0222
Fax:  301-480-3557
E-mail: hannah_peavy@NIH.GOV
Direct inquiries regarding fiscal matters to:
Mr. William Darby
Grants Operations Branch
National Heart, Lung, and Blood Institute
Two Rockledge Center, Suite 7128
6701 Rockledge Drive, MSC 7926
Bethesda, MD  20892-7926
Phone:  301-435-0177
Fax:  301-480-3310
This program is described in the Catalog of Federal Domestic
Assistance No. 93.837.  Awards are made under authorization
of the Public Health Service Act, Title IV, Part A (Public
Law 78-410, as amended by Public Law 99-158, 42 USC 241 and
285) and administered under PHS grants' policies and Federal
Regulations 42 CFR 52 and 45 CFR Part 74.  This program is
not subject to the intergovernmental review requirements of
Executive Order 12372 or Health Systems Agency review.
The PHS strongly encourages all grant and contract
recipients to provide a smoke-free workplace and promote the
non-use of all tobacco products.  In addition, Public Law
103-227, the Pro-Children Act of 1994, prohibits smoking in
certain facilities (or in some cases, any portion of a
facility) in which regular or routine education, library,
day care, health care or early childhood development
services are provided to children.  This is consistent with
the PHS mission to protect and advance the physical and
mental health of the American people.

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